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Viruses transmitted by Aedes mosquitoes are an increasingly important global cause of disease. Defining common determinants of host susceptibility to this large group of heterogenous pathogens is key for informing the rational design of panviral medicines. Infection of the vertebrate host with these viruses is enhanced by mosquito saliva, a complex mixture of salivary-gland-derived factors and microbiota. We show that the enhancement of infection by saliva was dependent on vascular function and was independent of most antisaliva immune responses, including salivary microbiota. Instead, the Aedes gene product sialokinin mediated the enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having a vertebrate-like tachykinin sequence and is absent from Anopheles mosquitoes , which are incompetent for most arthropod-borne viruses, whose saliva was not proviral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have the potential to limit disease severity following infection with Aedes -mosquito-borne viruses.

Analysis of chemokine receptor, and atypical chemokine receptor, expression is frequently hampered by the lack of availability of high‐quality antibodies and the species specificity of those that are available. We have previously described methodology utilizing Alexa‐Fluor‐labeled chemokine ligands as versatile reagents to detect receptor expression. Previously this has been limited to hematopoietic cells and methodology for assessing expression of receptors on stromal cells has been lacking. Among chemokine receptors, the ones most frequently expressed on stromal cells belong to the atypical chemokine receptor subfamily. These receptors do not signal in the classic sense in response to ligand but scavenge their ligands and degrade them and thus sculpt in vivo chemokine gradients. Here, we demonstrate the ability to use either intratracheal or intravenous, Alexa‐Fluor‐labeled chemokine administration to detect stromal cell populations expressing the atypical chemokine receptor ACKR2. Using this methodology, we demonstrate, for the first time, expression of ACKR2 on blood endothelial cells. This observation sets the lung aside from other tissues in which ACKR2 is exclusively expressed on lymphatic endothelial cells and suggest unique roles for ACKR2 in the pulmonary environment., Fluorescent‐CCL22 administration in vivo reveals pulmonary ACKR2 expression patterns. Intratracheal administration labels fibroblasts and some endothelial cells, while intravenous administration labels vascular endothelial cells. Our data provide the first evidence of ACKR2 expression on vascular endothelial cells. This is a versatile method applicable to analyzing other chemokine receptors in vivo.

SummaryViruses transmitted byAedesmosquitoes constitute an increasingly important global health burden. Defining common determinants of host susceptibility to this large group of heterogenous pathogens are key for informing the rational design of new pan-viral medicines. Infection of the vertebrate host with these viruses is enhanced by the presence of mosquito saliva, a complex mixture of salivary gland-derived factors and microbiota. We show that enhancement of infection by saliva was dependent on vascular function and was independent of most anti-saliva immune responses, including to salivary microbiota. Instead, theAedesgene product sialokinin mediated enhancement of virus infection through a rapid reduction in endothelial barrier integrity. Sialokinin is unique within the insect world as having vertebrate-like tachykinin sequence and is absent from non-vector competentAnophelesmosquitoes, whose saliva was not pro-viral and did not induce similar vascular permeability. Therapeutic strategies targeting sialokinin have potential to limit disease severity following infection withAedesmosquito-borne viruses.

Some free fatty acids derived from milk and vegetable oils are known to have potent antiviral and antibacterial properties. However, therapeutic applications of short- to medium-chain fatty acids are limited by physical characteristics such as immiscibility in aqueous solutions. We evaluated a novel proprietary formulation based on an emulsion of short-chain caprylic acid, ViroSAL, for its ability to inhibit a range of viral infections in vitro and in vivo. In vitro, ViroSAL inhibited the enveloped viruses Epstein–Barr, measles, herpes simplex, Zika and orf parapoxvirus, together with Ebola, Lassa, vesicular stomatitis and severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) pseudoviruses, in a concentration- and time-dependent manner. Evaluation of the components of ViroSAL revealed that caprylic acid was the main antiviral component; however, the ViroSAL formulation significantly inhibited viral entry compared with caprylic acid alone. In vivo, ViroSAL significantly inhibited Zika and Semliki Forest virus replication in mice following the inoculation of these viruses into mosquito bite sites. In agreement with studies investigating other free fatty acids, ViroSAL had no effect on norovirus, a non-enveloped virus, indicating that its mechanism of action may be surfactant disruption of the viral envelope. We have identified a novel antiviral formulation that is of great interest for the prevention and/or treatment of a broad range of enveloped viruses, particularly those of the skin and mucosal surfaces.

Mosquitoes can pass disease to humans when they bite, which includes infections caused by viruses, known as arboviruses. These viruses infect many millions of people each year and include viruses that cause diseases such as Zika, dengue and chikungunya. Most such infections are usually found in the tropics, but a changing climate and globalization means their range has spread at an alarming rate. Cumulatively these viruses are highly diverse at the genetic level and include up to 90 known human pathogens. It is hard to predict the nature, timing and location of these outbreaks. Furthermore, these viruses replicate to exceptionally high levels in the blood of infected patients. Thus, arbovirus infection constitutes an increasing concern for blood transfusion services. We suggest that it is important to identify common aspects of these infections to aid diagnosis, patient stratification and develop medicines that are applicable to multiple arbovirus infections. An important stage, common to all such infections, is the inoculation of virus at mosquito bites. We have recently shown that mosquito bite inflammation, which is common to all such infections, inadvertently enhances infection. In this study, we identified key aspects of mosquito bite inflammation that are important determinants of the subsequent systemic course and clinical outcome of infection. As such, host responses to mosquito saliva may prove to be a predictor of subsequent disease severity and consequently could also be useful in risk-stratifying blood products.

Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The abundance of genetically distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made the development of virus-specific treatments challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical TLR7 agonist suppressed both the local and subsequent systemic course of infection with a variety of arboviruses from the Alphavirus, Flavivirus, and Orthobunyavirus genera. Clinical outcome was improved in mice after infection with a model alphavirus. In the absence of treatment, antiviral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, stimulating the more populous skin-resident macrophages with a TLR7 agonist elicited protective responses in key cellular targets of virus that otherwise proficiently replicated virus. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have identified a putative new strategy for limiting disease after infection with a variety of genetically distinct arboviruses.

Macrophages are key regulators of developmental processes, including those involved in mammary gland development. We have previously demonstrated that the atypical chemokine receptor ACKR2 contributes to the control of ductal epithelial branching in the developing mammary gland by regulating macrophage dynamics. ACKR2 is a chemokine-scavenging receptor that mediates its effects through collaboration with inflammatory chemokine receptors (iCCRs). Here, we reveal reciprocal regulation of branching morphogenesis in the mammary gland, whereby stromal ACKR2 modulates levels of the shared ligand CCL7 to control the movement of a key population of CCR1-expressing macrophages to the ductal epithelium. In addition, oestrogen, which is essential for ductal elongation during puberty, upregulates CCR1 expression on macrophages. The age at which girls develop breasts is decreasing, which raises the risk of diseases including breast cancer. This study presents a previously unknown mechanism controlling the rate of mammary gland development during puberty and highlights potential therapeutic targets., Summary: The timing of pubertal mammary gland development is determined by availability of the chemokine CCL7, balanced by the scavenging receptor ACKR2 and the macrophage receptor CCR1.

Macrophages are key regulators of developmental processes, including those involved in mammary gland development. We previously demonstrated that the atypical chemokine receptor, ACKR2, contributes to control of ductal epithelial branching in the developing mammary gland by regulating macrophage dynamics. ACKR2 is a chemokine-scavenging receptor, which mediates its effects through collaboration with inflammatory chemokine receptors (iCCRs). Here we reveal that ACKR2, and the iCCR CCR1, reciprocally regulate branching morphogenesis in the mammary gland, whereby stromal ACKR2 modulates levels of the shared ligand CCL7 to control the movement of a key population of CCR1-expressing macrophages to the ductal epithelium. In addition estrogen, which is essential for ductal elongation during puberty, upregulates CCR1 expression on macrophages. The age at which girls develop breasts is decreasing, which raises the risk of diseases including breast cancer. This study presents a previously unknown mechanism controlling the rate of mammary gland development during puberty and highlights potential therapeutic targets.SummaryIn the mammary gland during puberty, availability of the chemokine CCL7 is controlled by a scavenging receptor ACKR2 and provides a key signal to macrophages which have the receptor CCR1. Together, this controls the timing of development.

Analysis of chemokine receptor, and atypical chemokine receptor, expression is frequently hampered by the lack of availability of high-quality antibodies and the species-specificity of those that are available. We have previously described methodology utilising Alexa-Fluor labelled chemokine ligands as versatile reagents to detect receptor expression. Previously this has been limited to haematopoietic cells and methodology for assessing expression of receptors on stromal cells has been lacking. Amongst chemokine receptors the ones most frequently expressed on stromal cells belong to the atypical chemokine receptor subfamily. These receptors do not signal in the classic sense in response to ligand but scavenge their ligands and degrade them and thus sculpt in vivo chemokine gradients. Here we demonstrate the ability to use either intratracheal, or intravenous, Alexa-Fluor labelled chemokine administration to detect stromal cell populations expressing the atypical chemokine receptor ACKR2. Using this methodology we demonstrate, for the first time, expression of ACKR2 on blood endothelial cells. This observation sets the lung aside from other tissues in which ACKR2 is exclusively expressed on lymphatic endothelial cells. In summary therefore we described a novel method for the in situ labelling of atypical chemokine receptor expressing cells appropriate for subsequent flow cytometric analysis. We propose that this methodology will work in a range of species and for a range of receptors and therefore will have significant versatility

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable.Rationalin silicodesign andin vitroscreens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic “swine” H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

Atypical chemokine receptor 2 (ACKR2) is a chemokine-scavenging receptor. ACKR2–/–embryos display a reduction in size of a novel, to our knowledge, embryonic skin macrophage population referred to as ‘intermediate’ cells. CC chemokine receptor 2 (CCR2)–/–embryos display an identical phenotype, indicating that these cells require CCR2 to enable them to populate embryonic skin. Further analysis revealed that ACKR2–/–embryos have higher circulating concentrations of the CCR2 ligand, CC ligand 2 (CCL2); thus, ACKR2 regulates intraembryonic CCL2 levels. We show that ACKR2 is strongly expressed by trophoblasts and that it blocks movement of inflammatory chemokines, such as CCL2, from the maternal decidua into the embryonic circulation. We propose that trophoblastic ACKR2 is responsible for ensuring chemokine compartmentalisation on the maternal decidua, without which chemokines enter the embryonic circulation, disrupting gradients essential for directed intraembryonic cell migration. Overall, therefore, we describe a novel, to our knowledge, molecular mechanism whereby maternal decidual chemokines can function in a compartmentalised fashion without interfering with intraembryonic leukocyte migration. These data suggest similar functions for other atypical chemokine receptors in the placenta and indicate that defects in such receptors may have unanticipated developmental consequences., A novel mechanism for molecular compartmentalisation in the placenta involves an atypical chemokine receptor that scavenges chemokines, blocking their drainage from the maternal face of the placenta into the embryo and thus protecting intraembryonic cellular migration processes.

Arthropod-borne viruses (arboviruses) are important human pathogens for which there are no specific antiviral medicines. The large number of genetically-distinct arbovirus species, coupled with the unpredictable nature of their outbreaks, has made developing virus-specific anti-viral medicines challenging. Instead, we have defined and targeted a key aspect of the host innate immune response to virus at the arthropod bite that is common to all arbovirus infections, potentially circumventing the need for virus-specific therapies at this site. Using mouse models and human skin explants, we identify innate immune responses by dermal macrophages in the skin as a key determinant of disease severity. Post-exposure treatment of the inoculation site by a topical innate immune agonist significantly suppressed both the local and subsequent systemic course of infection and improved clinical outcome in mice to infection with a variety of arboviruses from the Alphavirus, Flavivirus and Orthobunyavirus genuses. In the absence of treatment, anti-viral interferon expression to virus in the skin was restricted to dermal dendritic cells. In contrast, targeting the more populous skin-resident macrophages with an immune agonist elicited protective responses in key cellular targets of virus that otherwise replicated virus to high levels. By defining and targeting a key aspect of the innate immune response to virus at the mosquito bite site, we have shown that it is possible to improve outcome to infection by targeting pathways activated at the site of inoculation, and thereby identified a putative new strategy for limiting disease following infection with a variety of genetically-distinct arboviruses.One-sentence summaryWe demonstrate that activation of innate immune responses to arbovirus at the mosquito bite is a limiting factor for preventing efficient systemic dissemination of virus and that therapeutic targeting of skin-resident macrophages can have defining inhibitory effects on the later systemic course.

Aedes aegypti mosquitoes are responsible for transmitting\ud many medically important viruses such as\ud those that cause Zika and dengue. The inoculation\ud of viruses into mosquito bite sites is an important\ud and common stage of all mosquito-borne virus infections.\ud We show, using Semliki Forest virus and\ud Bunyamwera virus, that these viruses use this inflammatory\ud niche to aid their replication and dissemination\ud in vivo. Mosquito bites were characterized by\ud an edema that retained virus at the inoculation site\ud and an inflammatory influx of neutrophils that coordinated\ud a localized innate immune program that inadvertently\ud facilitated virus infection by encouraging\ud the entry and infection of virus-permissive myeloid\ud cells. Neutrophil depletion and therapeutic blockade\ud of inflammasome activity suppressed inflammation\ud and abrogated the ability of the bite to promote infection.\ud This study identifies facets of mosquito bite\ud inflammation that are important determinants of the\ud subsequent systemic course and clinical outcome\ud of virus infection.

Background:Skin in people with psoriasis has been comprehensively studied; uninvolved skin has abnormal gene expression. Less is known specifically about skin in PsA, the assumption being that it is identical to psoriasis. Chemokines and ACKR2 are among the upregulated genes in uninvolved psoriasis compared to healthy skin[1]. ACKR2 is a scavenging receptor of inflammatory CC chemokines and has been proposed as a regulator of cutaneous inflammation in psoriasis. It has not been studied in PsA.Objectives:To compare the transcriptome of PsA lesional, PsA uninvolved and healthy control skin and evaluate ACKR2 expression in PsA.Methods:Biopsies were taken from healthy control (HC) skin and paired lesional and uninvolved skin from patients with PsA. Libraries for bulk RNA sequencing were prepared from polyA selected RNA and sequenced on NovaSeq 6000. Sequencing data were analysed using Searchlight2. ACKR2 mRNA expression was validated by qPCR. RNAscope was used to localise ACKR2 expressing cells and sections were co-stained with podaplanin or stained in serial sections with CD45. Chemokine protein expression in skin was evaluated using Luminex technology.Results:Nine HC and 9 paired skin samples from patients with PsA were sequenced. The PsA skin lesions (PsA L) formed a distinct population in the transcriptomic principal component analysis (PCA) plot while HC and PsA uninvolved skin (PsA U) were overlapping. Only 15 genes were differentially expressed between HC and PsA U and none coded for chemokines. There were however significantly upregulated chemokines and receptors in PsA L. Unexpectely, ACKR2 was the 2ndmost upregulated chemokine receptor in PsA L with unchanged expression in PsA U compared with HC (PsA L vs HC log2fold 3.38, p.adj=9.51E-41; PsA L vs PsA U log2fold 3.58, p.adj=3.24E-45; PsA U vs HC log2fold -0.2, p.adj=0.732).The upregulation of ACKR2 in PsA L and unchanged expression in PsA U was confirmed by qPCR. RNAscope demonstrated strong expression of ACKR2 in the suprabasal layer of the epidermis in PsA L. In HC and PsA U, only occasional ACKR2 positive cells were seen in the epidermis. ACKR2 was expressed in lymphatic vessel walls but was not observed in CD45+ leukocytes.Provisional skin chemokine protein expression data showed poor correlation between mRNA levels and protein expression for the ACKR2 ligands CCL2, CCL3, CCL7, CCL8, CCL11, CCL13 and CCL22 in HC and PsA U, with negative correlation between ACKR2 mRNA expression and CCL2, CCL8 and CCL11 protein expression. In PsA L, chemokine mRNA correlated with protein expression, but protein expression of chemokine ligands did not correlate with ACKR2 expression.Conclusion:This data set shows expected upregulation of chemokines and their receptors in PsA L but relatively unchanged gene expression in PsA U, which contrasts to previous studies in psoriasis. Notably, this study demonstrates a strong upregulation of ACKR2 in keratinocytes in PsA L, with unchanged expression in PsA U. The RNA expression and preliminary protein data suggest that ACKR2 has little effect on the levels of its ligands in PsA skin lesions. However, this study may have missed local effects of ACKR2 in the epidermis.References:[1]Singh, M.D., et al.,Elevated expression of the chemokine-scavenging receptor D6 is associated with impaired lesion development in psoriasis.Am J Pathol, 2012.181(4): p. 1158-64.Acknowledgments:Funded by the Chief Scientist Office and a private donation to the University of Glasgow. Dr Sabarinadh Chilaka helped to prepare libraries for RNA sequencing.Disclosure of Interests:Hanna Johnsson: None declared, John Cole: None declared, Gillian Wilson: None declared, Marieke Pingen: None declared, Fiona McMonagle: None declared, Susan Holmes: None declared, Iain McInnes Grant/research support from: Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Janssen, and UCB, Consultant of: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Gilead, Janssen, Novartis, Pfizer, and UCB, Stefan Siebert Grant/research support from: BMS, Boehringer Ingelheim, Celgene, GlaxoSmithKline, Janssen, Novartis, Pfizer, UCB, Consultant of: AbbVie, Boehringer Ingelheim, Janssen, Novartis, Pfizer, UCB, Speakers bureau: AbbVie, Celgene, Janssen, Novartis, Gerard Graham: None declared

Chemokines have been shown to be essential players in a range of cancer contexts. In this study, we demonstrate that mice deficient in the atypical chemokine receptor Ackr2 display impaired development of metastasis in vivo in both cell line and spontaneous models. Further analysis reveals that this relates to increased expression of the chemokine receptor CCR2, specifically by KLRG1+ NK cells from the Ackr2−/− mice. This leads to increased recruitment of KLRG1+ NK cells to CCL2-expressing tumors and enhanced tumor killing. Together, these data indicate that Ackr2 limits the expression of CCR2 on NK cells and restricts their tumoricidal activity. Our data have important implications for our understanding of the roles for chemokines in the metastatic process and highlight Ackr2 and CCR2 as potentially manipulable therapeutic targets in metastasis.

Objectives: In similar to 10% of newly diagnosed HIV-1 patients, drug-resistant viral variants are detected. In such transmitted HIV-1 variants, the thymidine analogue mutation (TAM) M41L is frequently observed as a single resistance mutation and these viral variants often belong to phylogenetic transmission clusters. The presence of at least three TAMs, in particular patterns with M41L/L210W, impairs the efficacy of the extensively used drug tenofovir. We investigated whether the presence of a single M41L mutation at baseline influences the selection of resistance to tenofovir and emtricitabine in vitro and in vivo. Methods: The impact of M41L on the development of drug resistance to tenofovir and emtricitabine was determined by extensive in vitro selection experiments and investigation of the virological outcome of patients on a first-line regimen. Results: The presence of a single M41L mutation did not influence the selected mutational profile or the genetic barrier to resistance to tenofovir and/or emtricitabine during long-term in vitro selection experiments. In vivo, virological outcome of first-line regimens containing tenofovir and emtricitabine was comparable between patients diagnosed with HIV-1 harbouring M41L (n = 17, 16 were part of one transmission cluster) and WT virus (n = 248). Conclusions: Detection of a single M41L reverse transcriptase mutation at baseline did not influence the development of resistance in vitro or virological outcome on tenofovir-containing regimens in patients belonging to a large transmission cluster. Our results indicate that a high genetic barrier regimen may not be required when patients are diagnosed with HIV variants containing a single M41L mutation in reverse transcriptase.

Mosquito-borne infections are increasing in number and are spreading to new regions at an unprecedented rate. In particular, mosquito-transmitted viruses, such as those that cause Zika, dengue, West Nile encephalitis, and chikungunya, have become endemic or have caused dramatic epidemics in many parts of the world. Aedes and Culex mosquitoes are the main culprits, spreading infection when they bite. Importantly, mosquitoes do not act as simple conduits that passively transfer virus from one individual to another. Instead, host responses to mosquito-derived factors have an important influence on infection and disease, aiding replication and dissemination within the host. Here, we discuss the latest research developments regarding this fascinating interplay between mosquito, virus, and the mammalian host.

Background & Aim The immune system plays an important role in checking the development of tumours. While many inroads have been made into the role various immune cell types play within the context of cancer, further research is required to identify new cellular targets and improve anti-tumour therapeutics. Natural killer (NK) cells are involved in tumour rejection through direct cytotoxic mechanisms and the release of inflammatory mediators. As they are important in anti- tumour defence and amenable to ex vivo expansion, as shown in a number of clinical settings, NK cells are a candidate immunotherapy for cancer. Methods, Results & Conclusion Recent publications have shown that in mice lacking the atypical chemokine receptor ACKR2, NK cells have an increased expression of the chemokine receptor CCR2. ACKR2 KO animals demonstrate an increased protection from the development of metastatic lesions within the lungs compared to WT controls. Superior infiltration into the tumours by CCR2+ NK cells resulted in more efficient killing, and thus greater rejection of the newly-forming tumours. Here we provide evidence that CCR2+ NK cells can be isolated from the mouse using a GMP-compatible chemokine-based fluorescence sorting system, and that they are more capable than their CCR2- counterparts at inducing tumour cell death and reducing tumour burden following adoptive transfer in the B16F10 cancer model. In addition, we have generated preliminary data highlighting differential expression of CCR2 in human NK cells from healthy donor blood. Human NK cells were identified using CD56 expression and divided into two populations based on the intensity of the staining. Both CD56bright and CD56dim NK cells are capable of expressing CCR2. We have developed an adapted protocol for culturing and expanding NK cells, showing that expanded NK cells retain their phenotype and cytotoxicity over time. This is crucial to achieve a suitable cell number for potential therapeutic applications. We have performed multiple experiments to optimise the conditions for the maintenance of CCR2 expression and upregulation of the receptor on human NK cells. We are currently investigating whether CCR2+ human NK cells are equivalent to their murine counterparts, by assessing migratory capacity and cytotoxicity in vitro. Taking these data into account, we show that chemokine-sorted CCR2+ NK cells are a potentially viable immunotherapy for cancer.

Objectives The aim of the study was to gain more insight into the relationship between transmitted singletons found at HIV diagnosis by population sequencing and the possible presence of clinically relevant viral minorities containing additional resistance mutations. Methods We studied the viral quasispecies and therapy response in 10 individuals with transmitted single nucleoside reverse transcriptase inhibitor (NRTI)-related resistance mutations as detected by population sequencing. Results Ultra-deep pyrosequencing did not reveal additional drug-resistance mutations in nine of 10 patients. In these nine patients, no breakthrough with resistant viruses was observed despite the use of low genetic nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens in the majority of patients. Conclusions These data suggest that viral minority variants containing additional resistance mutations may be rare in patients with transmitted NRTI singletons in the Netherlands. Larger studies are required to confirm these findings and to determine the therapeutic consequences.

Background HIV-1-infected patients can be superinfected with additional HIV-1 variants. Therapy failure can be the consequence of an infection with a resistant strain. Methods A patient was diagnosed with a recent HIV-1 infection in April 2005 and subsequently clinically monitored. HIV-1 evolution was studied by population sequencing of the first 984 bases of the pol gene as well as 454 ultra-deep pyrosequencing (UDPS) of parts of the pol and env genes. Results The patient was diagnosed with a wild-type HIV-1 strain, but experienced rapid virological failure after initiating a non-nucleoside reverse transcriptase inhibitor (NNRTI)-based treatment regimen 3 years later. Population sequencing and UDPS revealed the presence of a second HIV-1 strain with a Y188L NNRTI resistance mutation in a sample obtained shortly prior to initiation of therapy. Phylogenetic analyses showed that the two HIV-1 strains were genetically distinct, providing evidence for superinfection. Conclusions The virological treatment failure in this patient was probably due to the superinfection with an NNRTI-resistant HIV-1 variant. Superinfection with drug-resistant strains can undermine HIV-1 treatment regimens selected on the basis of resistance testing at diagnosis. Patients, especially in high-risk groups, as well as their clinicians, should be aware of the risks and dangers of superinfections.

Background HIV–HBV-coinfected individuals who need to be treated only for their HBV infection have limited therapeutic options, since most approved anti-HBV agents have a risk of selecting for drug-resistant HIV mutants. In vivo data are inconclusive as to whether telbivudine (LdT) may exert antiviral effects against HIV. Thus, we investigated in further detail the antiviral activity and the biochemical properties of LdT against HIV-1. Methods To investigate the activity of LdT against HIV-1 in humans we analysed viral dynamics and genotypic and phenotypic resistance development in two HIV–HBV-coinfected individuals with no prior antiviral exposure. To investigate the activity of LdT against HIV-1 in vitro, LdT susceptibility for HIV-1 wild-type strains as well as drug-resistant strains was determined. Furthermore, we studied whether the 5′-triphosphate form of LdT (LdT-TP) can act as a substrate for wild-type HIV-1 RT. Results In the two patients studied, LdT treatment did not result in a significant decline of HIV-1 RNA load nor in selection of genotypic or phenotypic resistance in HIV-1 RT. In vitro virological analyses demonstrated that LdT had no activity (50% effective concentration >100 μM) against wild type HIV and drug-resistant variants. Biochemical analyses demonstrated that LdT-TP is not incorporated by wild-type HIV-1 RT. Conclusions Based on the in vivo and in vitro evidence obtained in this study, we conclude that LdT has no anti-HIV-1 activity and is currently the only selective anti-HBV agent among the five FDA-approved nucleoside/nucleotide analogues for treatment of HBV infections in HIV-infected individuals.

Background: In approximately 10% of newly diagnosed individuals in Europe, HIV-1 variants harboring transmitted drug resistance mutations (TDRM) are detected. For some TDRM it has been shown that they revert to wild type while other mutations persist in the absence of therapy. To understand the mechanisms explaining persistence we investigated the in vivo evolution of frequently transmitted HIV-1 variants and their impact on in vitro replicative capacity. Results: We selected 31 individuals infected with HIV-1 harboring frequently observed TDRM such as M41L or K103N in reverse transcriptase (RT) or M46L in protease. In all these samples, polymorphisms at non-TDRM positions were present at baseline (median protease: 5, RT: 6). Extensive analysis of viral evolution of protease and RT demonstrated that the majority of TDRM (51/55) persisted for at least a year and even up to eight years in the plasma. During follow-up only limited selection of additional polymorphisms was observed (median: 1). To investigate the impact of frequently observed TDRM on the replication capacity, mutant viruses were constructed with the most frequently encountered TDRM as site-directed mutants in the genetic background of the lab strain HXB2. In addition, viruses containing patient-derived protease or RT harboring similar TDRM were made. The replicative capacity of all viral variants was determined by infecting peripheral blood mononuclear cells and subsequently monitoring virus replication. The majority of site-directed mutations (M46I/M46L in protease and M41L, M41L + T215Y and K103N in RT) decreased viral replicative capacity; only protease mutation L90M did not hamper viral replication. Interestingly, most patient-derived viruses had a higher in vitro replicative capacity than the corresponding site-directed mutant viruses. Conclusions: We demonstrate limited in vivo evolution of protease and RT harbouring frequently observed TDRM in the plasma. This is in line with the high in vitro replication capacity of patient-derived viruses harbouring TDRM compared to site-directed mutant viruses harbouring TDRM. As site-directed mutant viruses have a lower replication capacity than the patient-derived viruses with similar mutational patterns, we propose that (baseline) polymorphisms function as compensatory mutations improving viral replication capacity.

Background: Different patterns of drug resistance are observed in treated and therapy naive HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naive population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns. As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals. Results: In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5(+) Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5(+) Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs. Conclusions: Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.

Transmitted resistant HIV may revert to wild-type in the absence of drug pressure due to reduced replication capacity (RC). We observed eight therapy-naive patients infected with HIV harbouring four mutations at nucleoside reverse transcriptase inhibitor (NRTI) resistance-related positions: M41L, T69S, L210E and T215S. If partial reverted resistance patterns like these are detected at baseline, concerns for more extensive resistance in the quasispecies often directs selection of first-line combination antiretroviral therapy (cART) towards more complex regimens.Genotypic resistance testing and phylogenetic analysis was performed using pol sequences of 400 therapy-naive patients and 1322 patients with at least one NRTI-related mutation. Reverse transcriptase (RT) genes were cloned into a reference strain and RC was investigated.Phylogenetic analysis showed that all eight patients are part of a transmission cluster (bootstrap value 92). The patients resided in three distinct geographical regions and were either homosexually or heterosexually infected. Prior negative serology and analysis of base ambiguity demonstrated circulation for at least 7 years. In vivo evolution showed a mixture with wild-type (T215S/T) in only one untreated patient more than 6 years after diagnosis. The reverted resistance pattern did not confer a substantial reduction in RC compared with wild-type, explaining its persistence in vivo and long-term circulation in the population. Four patients started cART; three of them received quadruple cART. All patients showed good virological and immunological response.Long-term circulation transcending distinct regions and transmission groups suggests reversion occurred in previous hosts in the transmission chain. Identification of clusters using epidemiological data and active-partner tracing may broaden therapeutic options in cases of transmitted resistance.

The aim of the study was to gain more insight into the relationship between transmitted singletons found at HIV diagnosis by population sequencing and the possible presence of clinically relevant viral minorities containing additional resistance mutations.We studied the viral quasispecies and therapy response in 10 individuals with transmitted single nucleoside reverse transcriptase inhibitor (NRTI)-related resistance mutations as detected by population sequencing.Ultra-deep pyrosequencing did not reveal additional drug-resistance mutations in nine of 10 patients. In these nine patients, no breakthrough with resistant viruses was observed despite the use of low genetic nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens in the majority of patients.These data suggest that viral minority variants containing additional resistance mutations may be rare in patients with transmitted NRTI singletons in the Netherlands. Larger studies are required to confirm these findings and to determine the therapeutic consequences.

Background Regulatory T cells (Treg) are important to maintain immune homeostasis. Presence of Treg correlates with a favourable disease course in juvenile idiopathic arthritis (JIA) patients. A distinction within the Treg population can be made between naturally occurring Treg (nTreg), which are derived from the thymus, and peripherally induced Treg (iTreg). Induction of Treg in the periphery is a promising way to modulate diseases like JIA, since it is easier to obtain high numbers of cells. However, the functionality of activation-induced CD4 + CD25 + FOXP3 + Treg has been highly debated in the last few years. Furthermore, in vivo induction of Treg by TCR or co-stimulation could induce a cytokine storm. Therefore, it is important to carefully monitor iTreg functionality. Objectives In this study, the authors tested whether human iTreg suppress immune responses in vitro, and in vivo in a humanised mouse model of xenogeneic graft versus host disease (x-GvHD). Methods CD4 + CD25 − T cells were isolated from human PBMC and cultured with aCD3/aCD28 with or without interleukin-2 and TGFβ to obtain iTreg. CD4 + CD25 high T cells were cultured with αCD3/αCD28, interleukin-2 and TGFβ to obtain nTreg. Supernatant was taken for Luminex analysis and cells were stained for Treg markers. Suppression assays were performed to determine suppressive capacity. RAG −/− γc −/− mice were sublethally irradiated and injected with clodronate liposomes to deplete phagocyting cells. Next, human PBMC were injected with or without iTreg or nTreg from the same human donor. Mice were scored for x-GvHD during 9 weeks. Results and conclusions The authors show here that induced Treg FOXP3 expression levels and suppressive capacity in vitro were comparable to nTreg. As expected, nTreg efficiently prevented acute x-GvHD. However, in contrast with nTreg, iTreg did not suppress disease. The results show that polyclonally induced Treg display no suppressive capacity in x-GvHD, due to a quick loss of FOXP3 in vivo. This underscores the importance to use humanised mouse models for validation of data regarding iTreg function obtained in in vitro assays, before proceeding to application in patients.

The encephalitic response to viral infection requires local chemokine production and the ensuing recruitment of immune and inflammatory leukocytes. Accordingly, chemokine receptors present themselves as plausible therapeutic targets for drugs aimed at limiting encephalitic responses. However, it remains unclear which chemokines are central to this process and whether leukocyte recruitment is important for limiting viral proliferation and survival in the brain or whether it is predominantly a driver of coincident inflammatory pathogenesis. Here we examine chemokine expression and leukocyte recruitment in the context of avirulent and virulent Semliki Forest virus (SFV) as well as West Nile virus infection and demonstrate rapid and robust expression of a variety of inflammatory CC and CXC chemokines in all models. On this basis, we define a chemokine axis involved in leukocyte recruitment to the encephalitic brain during SFV infection. CXCR3 is the most active; CCR2 is also active but less so, and CCR5 plays only a modest role in leukocyte recruitment. Importantly, inhibition of each of these receptors individually and the resulting suppression of leukocyte recruitment to the infected brain have no effect on viral titer or survival following infection with a virulent SFV strain. In contrast, simultaneous blockade of CXCR3 and CCR2 results in significantly reduced mortality in response to virulent SFV infection. In summary, therefore, our data provide an unprecedented level of insight into chemokine orchestration of leukocyte recruitment in viral encephalitis. Our data also highlight CXCR3 and CCR2 as possible therapeutic targets for limiting inflammatory damage in response to viral infection of the brain. IMPORTANCE Brain inflammation (encephalitis) in response to viral infection can lead to severe illness and even death. This therefore represents an important clinical problem and one that requires the development of new therapeutic approaches. Central to the pathogenesis of encephalitis is the recruitment of inflammatory leukocytes to the infected brain, a process driven by members of the chemokine family. Here we provide an in-depth analysis of the chemokines involved in leukocyte recruitment to the virally infected brain and demonstrate that simultaneous blockade of two of these receptors, namely, CXCR3 and CCR2, does not alter viral titers within the brain but markedly reduces inflammatory leukocyte recruitment and enhances survival in a murine model of lethal viral encephalitis. Our results therefore highlight chemokine receptors as plausible therapeutic targets in treating viral encephalitis.