The coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its deubiquitinating (DUB) and... Show moreThe coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its deubiquitinating (DUB) and deISGylating activities. To elucidate the role of these activities during SARS-CoV-2 infection, we introduced mutations that disrupt binding of PLpro to ubiquitin or ISG15. We identified several mutations that strongly reduced DUB activity of PLpro, without affecting viral polyprotein processing. In contrast, mutations that abrogated deISGylating activity also hampered viral polyprotein processing and when introduced into the virus these mutants were not viable. SARS-CoV-2 mutants exhibiting reduced DUB activity elicited a stronger interferon response in human lung cells. In a mouse model of severe disease, disruption of PLpro DUB activity did not affect lethality, virus replication, or innate immune responses in the lungs. This suggests that the DUB activity of SARS-CoV-2 PLpro is dispensable for virus replication and does not affect innate immune responses in vivo. Interestingly, the DUB mutant of SARS-CoV replicated to slightly lower titers in mice and elicited a diminished immune response early in infection, although lethality was unaffected. We previously showed that a MERS-CoV mutant deficient in DUB and deISGylating activity was strongly attenuated in mice. Here, we demonstrate that the role of PLpro DUB activity during infection can vary considerably between highly pathogenic coronaviruses. Therefore, careful considerations should be taken when developing pan-coronavirus antiviral strategies targeting PLpro. Show less
Bailey-Elkin, B.; Knaap, R.C.M.; Silva, A. de; Boekhoud, I.M.; Mous, S.; Vught, N. van; ... ; Mark, B.L. 2023
Deubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus... Show moreDeubiquitination of cellular substrates by viral proteases is a mechanism used to interfere with host cellular signaling processes, shared between members of the coronavirus- and arterivirus families. In the case of Arteriviruses, deubiquitinating and polyprotein processing activities are accomplished by the virus-encoded papain-like protease 2 (PLP2). Several studies have implicated the deubiquitinating activity of the porcine reproductive and respiratory syndrome virus (PRRSV) PLP2 in the downregulation of cellular interferon production, however to date, the only arterivirus PLP2 structure described is that of equine arteritis virus (EAV), a distantly related virus. Here we describe the first crystal structure of the PRRSV PLP2 domain both in the presence and absence of its ubiquitin substrate, which reveals unique structural differences in this viral domain compared to PLP2 from EAV. To probe the role of PRRSV PLP2 deubiquitinating activity in host immune evasion, we selectively removed this activity from the domain by mutagenesis and found that the viral domain could no longer downregulate cellular interferon production. Interestingly, unlike EAV, and also unlike the situation for MERS-CoV, we found that recombinant PRRSV carrying PLP2 DUBspecific mutations faces significant selective pressure to revert to wild-type virus in MARC145 cells, suggesting that the PLP2 DUB activity, which in PRRSV is present as three different versions of viral protein nsp2 expressed during infection, is critically important for PRRSV replication. Show less
Treffers, E.E.; Tas, A.; Scholte, F.E.M.; Ru, A.H. de; Snijder, E.J.; Veelen, P.A. van; Hemert, M.J. van 2023
Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host... Show moreChikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the similar to 3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381.Author summary Chikungunya virus (CHIKV) is an important human pathogen from the alphavirus family that has caused outbreaks in Africa, Asia and the Americas. CHIKV replication strongly depends on resources of the host cell, which undergoes many changes during infection. Here, we have used phosphoproteomics to obtain more insight into such changes, which revealed that CHIKV infection induces phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 regulates the elongation step of translation and its phosphorylation can strongly reduce protein synthesis. Previously, the nsP2 C-terminal domain was shown to reduce cellular transcription, which can prevent antiviral responses and promotes diversion of cellular resources to virus replication. Here, we describe that nsP2 also causes a shut-off of host-cell translation, which is mediated by the enzymatic activity of the nsP2 NTPase domain which acts through a previously unnoticed mechanism that involves cAMP, PKA and eEF2. Alphavirus infection or overexpression of nsP2 increased the cellular cAMP concentration, a second messenger that activates several signaling pathways, including the PKA and eEF2 kinases, of which the latter phosphorylates eEF2. Our observations highlight how alphaviruses modulate host cell metabolism on different levels and may account for poorly understood host shut-off effects observed in alphavirus-infected cells. Show less
Treffers, E.E.; Tas, A.; Scholte, F.E.M.; Ru, A.H. de; Snijder, E.J.; Veelen, P.A. van; Hemert, M.J. van 2023
Chikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host... Show moreChikungunya virus (CHIKV) is a reemerging alphavirus. Since 2005, it has infected millions of people during outbreaks in Africa, Asia, and South/Central America. CHIKV replication depends on host cell factors at many levels and is expected to have a profound effect on cellular physiology. To obtain more insight into host responses to infection, stable isotope labeling with amino acids in cell culture and liquid chromatography-tandem mass spectrometry were used to assess temporal changes in the cellular phosphoproteome during CHIKV infection. Among the ~3,000 unique phosphorylation sites analyzed, the largest change in phosphorylation status was measured on residue T56 of eukaryotic elongation factor 2 (eEF2), which showed a >50-fold increase at 8 and 12 h p.i. Infection with other alphaviruses (Semliki Forest, Sindbis and Venezuelan equine encephalitis virus (VEEV)) triggered a similarly strong eEF2 phosphorylation. Expression of a truncated form of CHIKV or VEEV nsP2, containing only the N-terminal and NTPase/helicase domains (nsP2-NTD-Hel), sufficed to induce eEF2 phosphorylation, which could be prevented by mutating key residues in the Walker A and B motifs of the NTPase domain. Alphavirus infection or expression of nsP2-NTD-Hel resulted in decreased cellular ATP levels and increased cAMP levels. This did not occur when catalytically inactive NTPase mutants were expressed. The wild-type nsP2-NTD-Hel inhibited cellular translation independent of the C-terminal nsP2 domain, which was previously implicated in directing the virus-induced host shut-off for Old World alphaviruses. We hypothesize that the alphavirus NTPase activates a cellular adenylyl cyclase resulting in increased cAMP levels, thus activating PKA and subsequently eukaryotic elongation factor 2 kinase. This in turn triggers eEF2 phosphorylation and translational inhibition. We conclude that the nsP2-driven increase of cAMP levels contributes to the alphavirus-induced shut-off of cellular protein synthesis that is shared between Old and New World alphaviruses. MS Data are available via ProteomeXchange with identifier PXD009381. Show less
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A... Show moreThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.Author summaryIn the last 3 years, it has become clear that emerging coronaviruses still pose a significant threat to human health. Despite the vaccines that are now available in many countries against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), vaccine scarcity or the inability to gain proper protection from immunization due to an immunocompromised status still leaves various people that will benefit from the administration of potent and safe antiviral agents. In this research, we have developed ubiquitin variants (UbVs), small proteins that resemble a natural component of human cells called ubiquitin, that are able to bind a part of the virus that is essential for its survival, with high affinity and selectivity. This way, these UbVs are able to inhibit the production of new viral particles after infection, thus preventing the virus from spreading from cell to cell and wreaking havoc on the body. Show less
Antibodies targeting the human malaria parasite Plasmodium falciparum circumsporozoite protein (PfCSP) can prevent infection and disease. PfCSP contains multiple central repeating NANP motifs; some... Show moreAntibodies targeting the human malaria parasite Plasmodium falciparum circumsporozoite protein (PfCSP) can prevent infection and disease. PfCSP contains multiple central repeating NANP motifs; some of the most potent anti-infective antibodies against malaria bind to these repeats. Multiple antibodies can bind the repeating epitopes concurrently by engaging into homotypic Fab-Fab interactions, which results in the ordering of the otherwise largely disordered central repeat into a spiral. Here, we characterize IGHV3-33/IGKV1-5-encoded monoclonal antibody (mAb) 850 elicited by immunization of transgenic mice with human immunoglobulin loci. mAb 850 binds repeating NANP motifs with picomolar affinity, potently inhibits Plasmodium falciparum (Pf) in vitro and, when passively administered in a mouse challenge model, reduces liver burden to a similar extent as some of the most potent anti-PfCSP mAbs yet described. Like other IGHV3-33/IGKV1-5-encoded anti-NANP antibodies, mAb 850 primarily utilizes its HCDR3 and germline-encoded aromatic residues to recognize its core NANP motif. Biophysical and cryo-electron microscopy analyses reveal that up to 19 copies of Fab 850 can bind the PfCSP repeat simultaneously, and extensive homotypic interactions are observed between densely-packed PfCSP-bound Fabs to indirectly improve affinity to the antigen. Together, our study expands on the molecular understanding of repeat-induced homotypic interactions in the B cell response against PfCSP for potently protective mAbs against Pf infection.Author summaryMalaria is a life-threatening disease caused by Pf parasites transmitted by infected mosquitoes. The surface of infectious Pf sporozoites is covered by PfCSP, which contains multiple, short amino-acid repeats in its central domain. Antibodies targeting the repeats have been shown to be capable of neutralizing the infection. Here, we describe monoclonal antibody (mAb) 850, which was isolated following immunization with a PfCSP repeat-containing antigen in a transgenic mouse model modified to express human Ig heavy and light chain variable regions. mAb 850 binds PfCSP repeats with high affinity and inhibits Pf sporozoites in in vitro and in vivo models of infection. Our molecular analyses reveal that similar to 19 copies of mAb 850 can simultaneously bind one molecule of PfCSP and induce a spiral-like conformation of the repeat. Extensive antibody-antibody contacts between mAbs result in formation of one of the highest-density mAb-CSP complexes yet described. Our findings improve our understanding of antibody-PfCSP interactions that mediate parasite inhibition. Show less
alpha-galactosidase (alpha-GAL) and alpha-N-acetylgalactosaminidase (alpha-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on... Show morealpha-galactosidase (alpha-GAL) and alpha-N-acetylgalactosaminidase (alpha-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important alpha-GAL and alpha-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni's alpha-GAL and alpha-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative alpha-GAL/alpha-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for alpha-GAL/alpha-NAGAL substrate cleavage. Both alpha-GAL and alpha-NAGAL enzymatic activities were higher in females compared to males (p<0.05; alpha-NAGAL > alpha-GAL), which was consistent with smp_089290's female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited alpha-NAGAL activity when compared to control worms (siLuc treated males, p<0.01; siLuc treated females, p<0.05). No significant reductions in alpha-GAL activities were observed in the same extracts. Despite this, decreases in alpha-NAGAL activities correlated with a significant inhibition in adult worm motility as well as in egg production. Programmed CRISPR/Cas9 editing of smp_089290 in adult worms confirmed the egg reduction phenotype. Based on these results, Smp_089290 was determined to act predominantly as an alpha-NAGAL (hereafter termed SmNAGAL) in schistosome parasites where it participates in coordinating movement and oviposition processes. Further characterisation of SmNAGAL and other functionally important glycosyl hydrolases may lead to the development of a novel anthelmintic class of compounds.Author summary Schistosomiasis is a parasitic disease caused by infection with blood flukes, which leads to acute and chronic pathology in millions of infected individuals located in deprived tropical and subtropical regions. Elucidating the function of schistosome genes has provided a clearer view on their roles in various molecular pathways, which are critical to successful parasitism. This information is invaluable when progressing novel drug and vaccine candidates. Here, we add to the existing knowledge of the Schistosoma mansoni parasitic glycan processing and modification machinery by functionally characterising a glycosyl hydrolase (S. mansoni alpha-N-acetylgalactosaminidase, SmNAGAL). We demonstrate that this protein is enzymatically active and important in coordinating parasite movement in adult male and female schistosomes. Additionally, we provide evidence that this protein regulates pathways associated with egg production in female schistosomes, which is responsible for inducing pathological reactions. Developing drugs that inhibit SmNAGAL enzymatic activity could provide a novel approach for controlling schistosomiasis. Show less
Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving as messenger RNAs that are translated into peptides. Here we describe circular RNAs generated by... Show moreCircular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving as messenger RNAs that are translated into peptides. Here we describe circular RNAs generated by human polyomaviruses (HPyVs), some of which encode variants of the previously described alternative large T antigen open reading frame (ALTO) protein. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N-6-methyladenosine (m(6)A) modified. The translation of MCPyV circALTOs into ALTO protein is negatively regulated by MCPyV-generated miRNAs in cultured cells. MCPyV ALTO expression increases transcription from some recombinant promoters in vitro and upregulates the expression of multiple genes previously implicated in MCPyV pathogenesis. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation in MCPyV-negative cells. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected in infected tissues and produces ALTO protein in cultured cells. Thus, human polyomavirus circRNAs are expressed in human tumors and infected tissues and express proteins that have the potential to modulate the infectious and tumorigenic properties of these viruses.Author summaryHuman polyomaviruses (HPyV) have been linked to diseases including Merkel cell carcinoma (MCC), a skin cancer caused by Merkel cell polyomavirus (MCPyV), and skin and hair eruptions caused by trichodysplasia spinulosa polyomavirus (TSPyV). We discover that HPyVs generate single-stranded, circular RNAs (circRNAs) in affected tissues. Both MCPyV and TSPyV generate circRNAs encompassing part of the early region (circALTOs) that can be translated to the alternative large T antigen open reading frame (ALTO) protein in cultured cells. Previously described microRNAs (miRNAs) generated by MCPyV have the ability to inhibit the expression of ALTO. MCPyV circALTOs are enriched in extracellular vesicles produced by MCC cell lines and cells that express circALTO. Finally, we discover that MCPyV ALTO protein enhances transcription from some recombinant promoters and promotes the expression of multiple host cell genes previously implicated in MCPyV pathogenesis. Thus, HPyVs generate circRNAs, including circALTOs that are translated to ALTO protein, and MCPyV ALTO has the ability to regulate transcription. Show less
Although a growing number of studies suggest interactions between Schistosoma parasites and viral infections, the effects of schistosome infections on the host response to viruses have not been... Show moreAlthough a growing number of studies suggest interactions between Schistosoma parasites and viral infections, the effects of schistosome infections on the host response to viruses have not been evaluated comprehensively. In this systematic review, we investigated how schistosomes impact incidence, virulence, and prevention of viral infections in humans and animals. We also evaluated immune effects of schistosomes in those coinfected with viruses. We screened 4,730 studies and included 103. Schistosomes may increase susceptibility to some viruses, including HIV and Kaposi's sarcoma-associated herpesvirus, and virulence of hepatitis B and C viruses. In contrast, schistosome infection may be protective in chronic HIV, Human T-cell Lymphotropic Virus-Type 1, and respiratory viruses, though further research is needed. Schistosome infections were consistently reported to impair immune responses to hepatitis B and possibly measles vaccines. Understanding the interplay between schistosomes and viruses has ramifications for anti-viral vaccination strategies and global control of viral infections.Author summary Many studies have described the effects of parasitic Schistosoma worm infections on the way that humans and animals respond to a variety of viral infections. Our goal was to evaluate, in a systematic manner, how having a schistosome parasitic infection affects a host's susceptibility to viral infections, the clinical disease course of viral infections, and prevention of viral infections by vaccines. We also assessed the effects of schistosome infection on the host immune response to viruses. We screened 4,730 studies for potential relevance and included 103 of them in this review. Overall, our analysis showed that schistosome infection impairs the host response to many viruses. This includes increasing host susceptibility to HIV and possibly Kaposi's sarcoma-associated herpesvirus, worsening the severity of clinical disease in hepatitis B and C infections, and decreasing immune responses to vaccines for hepatitis B and possibly measles. The studies that we analyzed also suggested that schistosome infection may protect the host against poor clinical outcomes from some viral infections including Human T-cell Lymphotropic Virus-Type 1, respiratory viruses, and chronic HIV. We discuss how these findings might be interpreted, and the additional research needed, in order to improve anti-viral vaccination strategies and control of viral infections globally. Show less
Author summaryArteriviruses are a rapidly expanding family of positive-stranded RNA viruses, which includes economically important veterinary pathogens like equine arteritis virus (EAV) and two... Show moreAuthor summaryArteriviruses are a rapidly expanding family of positive-stranded RNA viruses, which includes economically important veterinary pathogens like equine arteritis virus (EAV) and two species of porcine reproductive and respiratory syndrome virus (PRRSV-1 and PRRSV-2). In our previous studies, we uncovered an unprecedented arterivirus gene expression mechanism: a highly efficient -2 programmed ribosomal frameshift (PRF) that is controlled by an interaction of viral protein nsp1ss with specific RNA sequences and host poly(C) binding proteins. It is used by PRRSVs, and most other arteriviruses, to efficiently produce a previously unknown nonstructural protein variant, nsp2TF. In this study, we demonstrate that PRRSV nsp2TF interacts with the two major arteriviral envelope proteins, GP5 and M, whose heterodimerization in the secretory pathway is a key step in envelope protein trafficking and virus assembly. Our findings suggest that nsp2TF promotes arterivirus assembly by antagonizing the ubiquitination-dependent proteasomal degradation of GP5 and M proteins. This mechanism is based on the DUB activity of the PLP2 protease domain located within the N-terminal region of nsp2TF. To our knowledge, this is the first study to demonstrate that viruses can express a DUB that functions specifically to counteract the ubiquitination and degradation of key viral structural proteins.Arteriviruses are enveloped positive-strand RNA viruses that assemble and egress using the host cell's exocytic pathway. In previous studies, we demonstrated that most arteriviruses use a unique -2 ribosomal frameshifting mechanism to produce a C-terminally modified variant of their nonstructural protein 2 (nsp2). Like full-length nsp2, the N-terminal domain of this frameshift product, nsp2TF, contains a papain-like protease (PLP2) that has deubiquitinating (DUB) activity, in addition to its role in proteolytic processing of replicase polyproteins. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nsp2TF localizes to compartments of the exocytic pathway, specifically endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and Golgi complex. Here, we show that nsp2TF interacts with the two major viral envelope proteins, the GP5 glycoprotein and membrane (M) protein, which drive the key process of arterivirus assembly and budding. The PRRSV GP5 and M proteins were found to be poly-ubiquitinated, both in an expression system and in cells infected with an nsp2TF-deficient mutant virus. In contrast, ubiquitinated GP5 and M proteins did not accumulate in cells infected with the wild-type, nsp2TF-expressing virus. Further analysis implicated the DUB activity of the nsp2TF PLP2 domain in deconjugation of ubiquitin from GP5/M proteins, thus antagonizing proteasomal degradation of these key viral structural proteins. Our findings suggest that nsp2TF is targeted to the exocytic pathway to reduce proteasome-driven turnover of GP5/M proteins, thus promoting the formation of GP5-M dimers that are critical for arterivirus assembly. Show less
Species taxa are the units of taxonomy most suited to measure virus diversity, and they account for more than 70% of all virus taxa. Yet, as evidenced by the content of GenBank entries and... Show moreSpecies taxa are the units of taxonomy most suited to measure virus diversity, and they account for more than 70% of all virus taxa. Yet, as evidenced by the content of GenBank entries and illustrated by the recent literature on SARS-CoV-2, they are the most neglected taxa of virus research. To correct this disparity, we propose to make species taxa a first choice for communicating virus taxonomy in publications concerning viruses. We see it as a key step toward promoting research on diverse viruses, including pathogens, at this fundamental level of biology. Show less
Winkel, B.M.F.; Pelgrom, L.R.; Schuijlenburg, R. van; Baalbergen, E.; Ganesh, M.S.; Gerritsma, H.; ... ; Roestenberg, M. 2020
Author summary Malaria continues to be the deadliest parasitic disease worldwide, and an effective vaccine yielding sterile immunity does not yet exist. Attenuated parasites can induce sterile... Show moreAuthor summary Malaria continues to be the deadliest parasitic disease worldwide, and an effective vaccine yielding sterile immunity does not yet exist. Attenuated parasites can induce sterile protection in both human and rodent models for malaria, but these vaccines need to be administered directly into the bloodstream in order to convey protection; administration via the skin results in a much-reduced efficacy. We hypothesized this is caused by an early immune regulation initiated at the first site of contact with the immune system: the skin. However, the human skin stage of malaria has not been investigated to date. We used human antigen presenting cells as well as whole human skin explants to investigate (dermal) immune responses and found thatPlasmodiumsporozoites are able to suppress immune responses by inducing regulatory macrophages. Our study provides new insights in the mechanism of early immune regulation exploited byPlasmodiumparasites and can help to explain why intradermal vaccination using whole attenuated sporozoites results in reduced protection.Professional antigen-presenting cells (APCs), like macrophages (M phi s) and dendritic cells (DCs), are central players in the induction of natural and vaccine-induced immunity to malaria, yet very little is known about the interaction of SPZ with human APCs. Intradermal delivery of whole-sporozoite vaccines reduces their effectivity, possibly due to dermal immunoregulatory effects. Therefore, understanding these interactions could prove pivotal to malaria vaccination. We investigated human APC responses to recombinant circumsporozoite protein (recCSP), SPZ and anti-CSP opsonized SPZ both in monocyte derived MoDCs and MoM phi s. Both MoDCs and MoM phi s readily took up recCSP but did not change phenotype or function upon doing so. SPZ are preferentially phagocytosed by MoM phi s instead of DCs and phagocytosis greatly increased after opsonization. Subsequently MoM phi s show increased surface marker expression of activation markers as well as tolerogenic markers such as Programmed Death-Ligand 1 (PD-L1). Additionally they show reduced motility, produce interleukin 10 and suppressed interferon gamma (IFN gamma) production by antigen specific CD8(+)T cells. Importantly, we investigated phenotypic responses to SPZ in primary dermal APCs isolated from human skin explants, which respond similarly to their monocyte-derived counterparts. These findings are a first step in enhancing our understanding of pre-erythrocytic natural immunity and the pitfalls of intradermal vaccination-induced immunity. Show less
Karpiyevich, M.; Adjalley, S.; Mol, M.; Ascher, D.B.; Mason, B.; Noort, G.J.V. van; ... ; Artavanis-Tsakonas, K. 2019
Parasitic helminths evade, skew and dampen human immune responses through numerous mechanisms. Such effects will likely have consequences for HIV-1 transmission and disease progression. Here we... Show moreParasitic helminths evade, skew and dampen human immune responses through numerous mechanisms. Such effects will likely have consequences for HIV-1 transmission and disease progression. Here we analyzed the effects that soluble egg antigen (SEA) from Schistosoma mansoni had on modulating HIV-1 infection and cytokine/chemokine production in vitro. We determined that SEA, specifically through kappa-5, can potently bind to DC-SIGN and thereby blocks DC-SIGN mediated HIV-1 trans-infection (p<0.05) whilst not interfering with cis-infection. DCs exposed to SEA whilst maturing under Th2 promoting conditions, will upon co-culture with naive T-cells induce a T-cell population that was less susceptible to HIV-1 R5 infection (p<0.05) compared to DCs unexposed to SEA, whereas HIV-1 X4 virus infection was unaffected. This was not observed for DCs exposed to SEA while maturing under Th1 or Th1/Th2 (T-mix) promoting conditions. All T-cell populations induced by SEA exposed DCs demonstrate a reduced capacity to produce IFN-gamma and MIP-1 beta. The infection profile of T-cells infected with HIV-1 R5 was not associated with down-modulation of CCR5 cell surface expression. We further show that DCs maturing under T-mix conditions exposed to plant recombinant omega-1 protein (r omega-1), which demonstrates similar functions to natural omega-1, induced T-cell populations that were less sensitive for HIV-1 R5 infection (p<0.05), but not for X4 virus infection. This inhibition associated again with a reduction in IFN-gamma and MIP-1 beta expression, but additionally correlated with reduced CCR5 expression. We have shown that SEA parasite antigens and more specifically r omega-1 can modulate HIV-1 infectivity with the potential to influence disease course in co-infected individuals.Author summary Parasitic helminths have developed a number of strategies to evade, skew and dampen human immune responses. Such effects will likely have consequences for HIV-1 transmission and disease progression. Here we analyzed the effect that soluble egg antigen (SEA) from Schistosoma mansoni had on HIV-1 infection in vitro. We determined that SEA, through kappa-5, can potently block DC-SIGN mediated HIV-1 trans-infection of CD4(+) T-lymphocytes, but not block cis-infection. Dendritic cells (DC) exposed to SEA during maturation under Th2 skewing conditions, induce T-cell populations that are less susceptible to HIV-1 R5 infection compared to cells induced by unexposed DCs. HIV-1 X4 infection was unaffected. This restricted infection profile was not associated with down-modulation of CCR5 surface expression or observed differences in cytokine/chemokine production. Using recombinant omega-1, an abundant component of SEA, HIV-1 R5 infection was similarly inhibited with no effect on HIV-1 X4 infection levels. Hence SEA possesses antigens, namely omega-1, that can modulate HIV-1 infection and potentially influence disease course in co-infected individuals. Show less
Mycobacterial pathogens are the causative agents of chronic infectious diseases like tuberculosisand leprosy. Autophagy has recently emerged as an innate mechanism for defense against these... Show moreMycobacterial pathogens are the causative agents of chronic infectious diseases like tuberculosisand leprosy. Autophagy has recently emerged as an innate mechanism for defense against these intracellular pathogens. In vitro studies have shown that mycobacteria escaping from phagosomes into the cytosol are ubiquitinated and targeted by selective autophagy receptors. However, there is currently no in vivo evidence for the role of selective autophagy receptors in defense against mycobacteria, and the importance of autophagy in control of mycobacterial diseases remains controversial. Here we have used Mycobacterium marinum (Mm), which causes a tuberculosis-like disease in zebrafish, to investigate the function of two selective autophagy receptors, Optineurin (Optn) and SQSTM1 (p62), in host defense against a mycobacterial pathogen. To visualize the autophagy response to Mm in vivo, optn and p62 zebrafish mutant lines were generated in the background of a GFP-Lc3 autophagy reporter line. We found that loss-of-function mutation of optn or p62 reduces autophagic targeting of Mm, and increases susceptibility of the zebrafish host to Mm infection. Transient knockdown studies confirmed the requirement of both selective autophagy receptors for host resistance against Mm infection. For gain-of-function analysis, we overexpressed optn or p62 by mRNA injection and found this to increase the levels of GFP-Lc3 puncta in association with Mm and to reduce the Mm infection burden. Taken together, our results demonstrate that both Optn and p62 are required for autophagic host defense against mycobacterial infection and support that protection against tuberculosis disease may be achieved by therapeutic strategies that enhance selective autophagy. Show less
