HBHA-Induced Polycytotoxic CD4+ T Lymphocytes Are Associated with the Control of Mycobacterium tuberculosis Infection in Humans.

Heparin-binding hemagglutinin (HBHA), a surface protein of Mycobacterium tuberculosis, is an attractive vaccine candidate and marker of protective immunity against tuberculosis, although the mechanisms underlying this protective immunity are not fully understood. Comparisons of the immune responses of latently M. tuberculosis-infected (LTBI) subjects to those of patients with active tuberculosis (aTB) may help to identify surrogate markers of protection, as LTBI subjects are most often lifelong protected against the disease. HBHA was shown to induce strong Th1 responses and cytotoxic CD8+ responses in LTBI subjects, but additional mechanisms of control of M. tuberculosis infection remain to be identified. In this study, using HBHA-induced blast formation as a readout of specific T lymphocyte activation, we report the presence in M. tuberculosis-infected subjects of HBHA-induced CD4+ T cell blasts that degranulate, as measured by surface capture of CD107a. This suggests the induction by HBHA of a CD4+ T cell subset with cytolytic function, and as nearly half of these cells also contained IFN-γ, they had both Th1 and cytotoxic characteristics. We further identified a CD4+ T lymphocyte subset producing IFN-γ together with a combination of mediators of cytotoxicity, i.e., perforin, granzymes, and granulysin, and we called them polycytotoxic CD4+ T lymphocytes. Interestingly, whereas purified protein derivative induced such cells in both LTBI subjects and patients with aTB, HBHA-specific polycytotoxic CD4+ T lymphocytes were detected in LTBI subjects and not in patients with pulmonary aTB. To our knowledge, we thus identified a new HBHA-induced CD4+ T cell subset that may contribute to the control of M. tuberculosis infection.

Adjuvant effect of the human metapneumovirus (HMPV) matrix protein in HMPV subunit vaccines.

The human metapneumovirus (HMPV) fusion (F) protein is the most immunodominant protein, yet subunit vaccines containing only this protein do not confer complete protection. The HMPV matrix (M) protein induces the maturation of antigen-presenting cells in vitro. The inclusion of the M protein into an F protein subunit vaccine might therefore provide an adjuvant effect. We administered the F protein twice intramuscularly, adjuvanted with alum, the M protein or both, to BALB/c mice at 3 week intervals. Three weeks after the boost, mice were infected with HMPV and monitored for 14 days. At day 5 post-challenge, pulmonary viral titres, histopathology and cytokine levels were analysed. Mice immunized with F+alum and F+M+alum generated significantly more neutralizing antibodies than mice immunized with F only [titres of 47 ± 7 (P<0.01) and 147 ± 13 (P<0.001) versus 17 ± 2]. Unlike F only [1.6 ± 0.5 × 10(3) TCID50 (g lung)(-1)], pulmonary viral titres in mice immunized with F+M and F+M+alum were undetectable. Mice immunized with F+M presented the most important reduction in pulmonary inflammation and the lowest T-helper Th2/Th1 cytokine ratio. In conclusion, addition of the HMPV-M protein to an F protein-based vaccine modulated both humoral and cellular immune responses to subsequent infection, thereby increasing the protection conferred by the vaccine.

Differential expression of maturation and activation markers on NK cells in patients with active and latent tuberculosis.

NK cells were recently suggested to be important for the initial control of M. tuberculosis infection. The phenotypes of the 3 main NK blood subsets, CD56bright , CD56dim , and CD56neg cells, were characterized by flow cytometry in a cohort of 81 prospectively enrolled subjects (21 untreated patients with active tuberculosis -aTB-, 35 latently TB infected -LTBI- subjects, and 25 non-infected controls), using 9 different mAbs added to whole blood. Compared to LTBI subjects, patients with aTB had lower proportions of total NK cells, lower proportions and numbers of CD56neg cells expressing early maturation markers (CD161, NKp30, NKp46), but higher density of NKp30 and NKp46 expression on both CD56neg and CD56dim subsets, associated with higher expression of granzymes A/B. They also had higher proportions of activated CD69pos cells within all 3 NK cell subsets and, the percentage of CD69pos CD56dim cells among CD69pos and/or NKG2Cpos NK cells was identified as a potential biomarker to discriminate aTB from LTBI. LTBI subjects were in contrast characterized by higher expression of late maturation markers (CD57, KIR molecules) on the CD56neg subset, by higher proportions of NKG2Cpos KIRpos CD56dim NK cells, and by higher in vitro IFN-γ production than patients with aTB. Thus, the in-depth phenotypic characterization of blood NK cell subsets provides new insights on possible functional modifications and the potential role of NK cells in the control of M. tuberculosis infection in humans.

Long-term impairment of Streptococcus pneumoniae lung clearance is observed after initial infection with influenza A virus but not human metapneumovirus in mice.

Human metapneumovirus (hMPV) is a paramyxovirus responsible for respiratory tract infections in humans. Our objective was to investigate whether hMPV could predispose to long-term bacterial susceptibility, such as previously observed with influenza viruses. BALB/c mice were infected with hMPV or influenza A and, 14 days following viral infection, challenged with Streptococcus pneumoniae. Only mice previously infected with influenza A demonstrated an 8% weight loss of their body weight 72 h following S. pneumoniae infection, which correlated with an enhanced lung bacterial replication of >7 log(10) compared with pneumococcus infection alone. This enhanced bacterial replication was not related to altered macrophage or neutrophil recruitment or deficient production of critical cytokines. However, bacterial challenge induced the production of gamma interferon in bronchoalveolar lavages of influenza-infected mice, but not in those of hMPV-infected animals. In conclusion, hMPV does not cause long-term impairment of pneumococcus lung clearance, in contrast to influenza A virus.

Nearly Complete Genome Sequences of Two Bluetongue Viruses Isolated during the 2020 Outbreak in the Grand Duchy of Luxembourg.

Bluetongue is one of the major diseases of ruminants listed by the World Organisation for Animal Health. Bluetongue virus serotype 8 (BTV-8) has been considered enzootic in France since 2018. Here, we report the nearly complete genome sequences of two BTV-8 isolates from the 2020 outbreak in the Grand Duchy of Luxembourg.

Novel Homozygous Inactivating Mutation in the PCSK1 Gene in an Infant with Congenital Malabsorptive Diarrhea.

Proprotein convertase 1/3 (PC1/3), encoded by the PCSK1 gene, is expressed in neuronal and (entero)endocrine cell types, where it cleaves and hence activates a number of protein precursors that play a key role in energy homeostasis. Loss-of-function mutations in PCSK1 cause a recessive complex endocrinopathy characterized by malabsorptive diarrhea and early-onset obesity. Despite the fact that neonatal malabsorptive diarrhea is observed in all patients, it has remained understudied. The aim of this study was to investigate the enteroendocrine pathologies in a male patient with congenital PCSK1 deficiency carrying the novel homozygous c.1034A>C (p.E345A) mutation. This patient developed malabsorptive diarrhea and metabolic acidosis within the first week of life, but rapid weight gain was observed after total parenteral nutrition, and he displayed high proinsulin levels and low adrenocorticotropin. In vitro analysis showed that the p.E345A mutation in PC1/3 resulted in a (near) normal autocatalytic proPC1/3 processing and only partially impaired PC1/3 secretion, but the processing of a substrate in trans was completely blocked. Immunohistochemical staining did not reveal changes in the proGIP/GIP and proglucagon/GLP-1 ratio in colonic tissue. Hence, we report a novel PCSK1 deficient patient who, despite neonatal malabsorptive diarrhea, showed a normal morphology in the small intestine.

Comparative Evaluation of Lumpy Skin Disease Virus-Based Live Attenuated Vaccines.

Vaccines form the cornerstone of any control, eradication and preventative strategy and this is no different for lumpy skin disease. However, the usefulness of a vaccine is determined by a multiplicity of factors which include stability, efficiency, safety and ease of use, to name a few. Although the vaccination campaign in the Balkans against lumpy skin disease virus (LSDV) was successful and has been implemented with success in the past in other countries, data of vaccine failure have also been reported. It was therefore the purpose of this study to compare five homologous live attenuated LSDV vaccines (LSDV LAV) in a standardized setting. All five LSDV LAVs studied were able to protect against a challenge with virulent LSDV. Aside from small differences in serological responses, important differences were seen in side effects such as a local reaction and a Neethling response upon vaccination between the analyzed vaccines. These observations can have important implications in the applicability in the field for some of these LSDV LAVs.

Detection of Clinical and Subclinical Lumpy Skin Disease Using Ear Notch Testing and Skin Biopsies.

Lumpy skin disease (LSD) diagnosis is primarily based on clinical surveillance complemented by PCR of lesion crusts or nodule biopsies. Since LSD can be subclinical, the sensitivity of clinical surveillance could be lower than expected. Furthermore, real-time PCR for the detection of LSD viral DNA in blood samples from subclinical animals is only intermittently positive. Therefore, this study aimed to investigate an acceptable, easily applicable and more sensitive testing method for the detection of clinical and subclinical LSD. An animal experiment was conducted to investigate ear notches and biopsies from unaffected skin taken from the neck and dorsal back as alternatives to blood samples. It was concluded that for early LSD confirmation, normal skin biopsies and ear notches are less fit for purpose, as LSDV DNA is only detectable in these samples several days after it is detectable in blood samples. On the other hand, blood samples are less advisable for the detection of subclinical animals, while ear notches and biopsies were positive for LSD viral DNA in all subclinically infected animals by 16 days post infection. In conclusion, ear notches could be used for surveillance to detect subclinical animals after removing the clinical animals from a herd, to regain trade by substantiating the freedom of disease or to support research on LSDV transmission from subclinical animals.

The Importance of Quality Control of LSDV Live Attenuated Vaccines for Its Safe Application in the Field.

Vaccination is an effective approach to prevent, control and eradicate diseases, including lumpy skin disease (LSD). One of the measures to address farmer hesitation to vaccinate is guaranteeing the quality of vaccine batches. The purpose of this study was to demonstrate the importance of a quality procedure via the evaluation of the LSD vaccine, Lumpivax (Kevevapi). The initial PCR screening revealed the presence of wild type LSD virus (LSDV) and goatpox virus (GTPV), in addition to vaccine LSDV. New phylogenetic PCRs were developed to characterize in detail the genomic content and a vaccination/challenge trial was conducted to evaluate the impact on efficacy and diagnostics. The characterization confirmed the presence of LSDV wild-, vaccine- and GTPV-like sequences in the vaccine vial and also in samples taken from the vaccinated animals. The analysis was also suggestive for the presence of GTPV-LSDV (vaccine/wild) recombinants. In addition, the LSDV status of some of the animal samples was greatly influenced by the differentiating real-PCR used and could result in misinterpretation. Although the vaccine was clinically protective, the viral genomic content of the vaccine (being it multiple Capripox viruses and/or recombinants) and the impact on the diagnostics casts serious doubts of its use in the field.

Coding-Complete Sequences of Recombinant Lumpy Skin Disease Viruses Collected in 2020 from Four Outbreaks in Northern Vietnam.

Lumpy skin disease virus (LSDV) causes a severe, systemic, and economically important disease in cattle. Here, we report coding-complete sequences of recombinant LSDVs from four outbreaks in October and November 2020 in northeastern Vietnam.

Prolyl endopeptidase-like is a (thio)esterase involved in mitochondrial respiratory chain function.

Deficiency of the serine hydrolase prolyl endopeptidase-like (PREPL) causes a recessive metabolic disorder characterized by neonatal hypotonia, feeding difficulties, and growth hormone deficiency. The pathophysiology of PREPL deficiency and the physiological substrates of PREPL remain largely unknown. In this study, we connect PREPL with mitochondrial gene expression and oxidative phosphorylation by analyzing its protein interactors. We demonstrate that the long PREPLL isoform localizes to mitochondria, whereas PREPLS remains cytosolic. Prepl KO mice showed reduced mitochondrial complex activities and disrupted mitochondrial gene expression. Furthermore, mitochondrial ultrastructure was abnormal in a PREPL-deficient patient and Prepl KO mice. In addition, we reveal that PREPL has (thio)esterase activity and inhibition of PREPL by Palmostatin M suggests a depalmitoylating function. We subsequently determined the crystal structure of PREPL, thereby providing insight into the mechanism of action. Taken together, PREPL is a (thio)esterase rather than a peptidase and PREPLL is involved in mitochondrial homeostasis.

An Immunoperoxidase Monolayer Assay (IPMA) for the detection of lumpy skin disease antibodies.

During this study a new Immunoperoxidase Monolayer Assay (IPMA) was developed for the detection of antibodies against lumpy skin disease virus (LSDV) in an easy and low tech setting. Using two dilutions (1:50 and 1:300) in a duplicate format, the test was shown to be highly sensitive, specific and repeatable. In comparison to the VNT and a commercial ELISA, the LSDV-IPMA was able to detect the LSDV antibodies earlier in infected, vaccinated and vaccinated/infected animals. The assay is very flexible as it can be easily adapted for the detection of sheeppox or goatpox antibodies and it can be scaled-up to handle medium size sample sets by preparing the IPMA plates in advance. These plates are safe and can be handled in low biosafety level labs.

Complete Coding Sequence of a Lumpy Skin Disease Virus from an Outbreak in Bulgaria in 2016.

Lumpy skin disease (LSD) is an emerging cattle disease with serious economic consequences. We report the complete coding sequence of LSD virus 210LSD-249/BUL/16, detected in a blood sample from a diseased cow during an outbreak in Bulgaria (Kabile Village, Yambol Region) in June 2016.

Complete Coding Sequence of a Lumpy Skin Disease Virus Strain Isolated during the 2016 Outbreak in Kazakhstan.

Lumpy skin disease virus (LSDV) causes an economically important disease in cattle. Here, we report the complete coding sequence of the LSDV isolate Kubash/KAZ/16, detected in a clinical sample from an infected cow from the outbreak reported on 7 July 2016 in Kazakhstan (Atyrau Region).

CD4 mimetic miniproteins: potent anti-HIV compounds with promising activity as microbicides.

OBJECTIVES: The antiviral activity of CD4 miniproteins was evaluated as potential HIV microbicides, using relevant in vitro models. METHODS: Compounds were tested in a single-cycle HIV-1 pseudovirus assay and against replication competent HIV-1 in co-cultures of monocyte-derived dendritic cells (MO-DC) and CD4+ T cells. Cytotoxic activity was evaluated in an MTT assay. RESULTS: Monomeric miniproteins (M47 and M48) showed 50% effective concentration (EC(50)) values of 79-105 nM against a subtype B, CCR5 co-receptor-using Ba-L pseudovirus. Higher activity was found for the dimeric miniproteins M48D30, M48D50 and M48D100 (EC(50) between 15 and 30 nM), in contrast to the tetrameric miniproteins M48T30, M48T50 and M48T100 (EC(50) between 107 and 377 nM). The hetero-bivalent miniprotein M48-Hep and miniproteins that targeted the Phe-43 cavity on gp120 (M48-U1, M48-U2 and M48-U3) were highly active, with EC(50) values as low as 2 nM for M48-U1. All miniproteins showed high activity against CCR5 or CXCR4 co-receptor-using subtype B and CRF-01_A/E pseudoviruses. Many early M48-based compounds were much less active against subtype C pseudoviruses, whereas M48-U compounds that targeted the Phe-43 cavity were very active against all pseudoviruses, including subtype C. In MO-DC/CD4+ T cell co-cultures with replication-competent HIV-1 Ba-L, EC(50) values ranged between 13 and 1719 nM depending on the miniprotein, with M48-U1, M48-U2 and M48-U3 again being the most potent. Importantly, the latter compounds completely prevented viral replication by treating the cultures from 2 h before until 24 h after infection, at non-toxic concentrations of 66-6564 nM. CONCLUSIONS: These novel CD4 miniproteins might constitute a promising class of HIV microbicides.

Antiviral compounds show enhanced activity in HIV-1 single cycle pseudovirus assays as compared to classical PBMC assays.

HIV-1 Env pseudotyped viruses (PV) are an attractive tool for studying the antiviral activities of compounds interfering with virus entry into a target cell. To investigate whether results obtained in PV assays are relevant biologically, the antiviral activity of 6 reference compounds was compared on 5 virus isolates of different clades using three assays: (1) replicating virus in peripheral blood mononuclear cells (PBMCs), (2) PV in CD4 and CCR5- or CXCR4 co-receptor expressing Ghost cells, and (3) PV in PBMCs. A significant linear relationship was found between both single-cycle PV assays (P<0.0001, R2=0.75). Moreover, both assays showed enhanced sensitivity to the antiretrovirals tested (P=0.013 and 0.015, respectively) as compared to the PBMC assay with replication-competent virus. Most importantly, results from the latter assay could be predicted significantly from both PV assays, in which either Ghost target cells (P<0.0001, R2=0.61) or PBMCs (P<0.0001, R2=0.55) were used. The usefulness of the PV assay was demonstrated further by investigating the impact of the HIV-1 Env subtype on the antiviral activity of five new compounds derived from the entry inhibitor BMS806.

Blood tolerogenic monocytes and low proportions of dendritic cell subpopulations are hallmarks of human tuberculosis.

BACKGROUND: The immune mechanisms underlying the pathogenesis of tuberculosis (TB) need better understanding to improve TB management, as the disease still causes more than 1.5 million deaths annually. This study tested the hypothesis that a modulation of the proportions or activation status of APC during Mycobacterium tuberculosis infection may impact on the course of the disease. PROCEDURE: Proportions of circulating APC subsets and the expression of stimulatory (CD86), inhibitory (ILT-3, ILT-4, ILT-7), or apoptosis-inducing (PDL-1, PDL-2) molecules were analyzed in 2 independent cohorts, on blood monocytes and dendritic cell (DC) subsets from patients with active or latent TB infection (aTB /LTBI) and from uninfected subjects. RESULTS: Higher proportions of classical CD14+ CD16- and intermediate CD14+ CD16+ monocytes, and lower proportions of plasmacytoid DC (pDC) and type 2 myeloid DC were observed in the blood from untreated patients with aTB compared with those with LTBI and with healthy subjects, with an early normalization of the proportions of pDC during treatment. In addition, monocytes from M. tuberculosis-infected subjects expressed higher levels of ILT-3, ILT-4, and PDL-1 compared with healthy controls, these differences being more important for patients with aTB than for those with LTBI. CONCLUSIONS: These results confirm the hypothesis of a modulation of the proportions and activation status of APC during M. tuberculosis infection and suggest that these cells could play a role in driving the course of M. tuberculosis infection.

5-HTR2A and 5-HTR3A but not 5-HTR1A antagonism impairs the cross-modal reactivation of deprived visual cortex in adulthood.

Visual cortical areas show enhanced tactile responses in blind individuals, resulting in improved behavioral performance. Induction of unilateral vision loss in adult mice, by monocular enucleation (ME), is a validated model for such cross-modal brain plasticity. A delayed whisker-driven take-over of the medial monocular zone of the visual cortex is preceded by so-called unimodal plasticity, involving the potentiation of the spared-eye inputs in the binocular cortical territory. Full reactivation of the sensory-deprived contralateral visual cortex is accomplished by 7 weeks post-injury. Serotonin (5-HT) is known to modulate sensory information processing and integration, but its impact on cortical reorganization after sensory loss, remains largely unexplored. To address this issue, we assessed the involvement of 5-HT in ME-induced cross-modal plasticity and the 5-HT receptor (5-HTR) subtype used. We first focused on establishing the impact of ME on the total 5-HT concentration measured in the visual cortex and in the somatosensory barrel field. Next, the changes in expression as a function of post-ME recovery time of the monoamine transporter 2 (vMAT2), which loads 5-HT into presynaptic vesicles, and of the 5-HTR1A and 5-HTR3A were assessed, in order to link these temporal expression profiles to the different types of cortical plasticity induced by ME. In order to accurately pinpoint which 5-HTR exactly mediates ME-induced cross-modal plasticity, we pharmacologically antagonized the 5-HTR1A, 5-HTR2A and 5-HTR3A subtypes. This study reveals brain region-specific alterations in total 5-HT concentration, time-dependent modulations in vMAT2, 5-HTR1A and 5-HTR3A protein expression and 5-HTR antagonist-specific effects on the post-ME plasticity phenomena. Together, our results confirm a role for 5-HTR1A in the early phase of binocular visual cortex plasticity and suggest an involvement of 5-HTR2A and 5-HTR3A but not 5-HTR1A during the late cross-modal recruitment of the medial monocular visual cortex. These insights contribute to the general understanding of 5-HT function in cortical plasticity and may encourage the search for improved rehabilitation strategies to compensate for sensory loss.

Effect of in vitro syncytium formation on the severity of human metapneumovirus disease in a murine model.

Human metapneumovirus (HMPV) is an important cause of acute respiratory tract infections (ARTI) in children, elderly individuals and immunocompromised patients. In vitro, different HMPV strains can induce variable cytopathic effects ranging from large multinucleated syncytia to focal cell rounding. In this study, we investigated the impact of different in vitro phenotypes of two HMPV strains on viral replication and disease severity in a BALB/c mouse model. We first generated two recombinant GFP-expressing HMPV viruses: C-85473, a syncytium-inducing strain (rC-85473) belonging to the A1 subtype and CAN98-75, a focal cell rounding-inducing strain (rCAN98-75) of the B2 subtype. We subsequently exchanged the F genes of both strains to create the chimeric viruses rC-85473_F and rCAN98-75_F. We demonstrated that the F protein was the sole protein responsible for the syncytium phenotype and that viruses carrying a syncytium-inducing F protein replicated to significantly higher titers in vitro. In vivo, however, the virulence and replicative capacity of the different HMPV strains did not appear to be solely dependent on the F gene but also on the viral background, with the strains containing the C-85473 background inducing more weight loss as well as increased lung viral titers, pro-inflammatory cytokines and inflammation than strains containing the CAN98-75 background. In conclusion, the F protein is the main determinant of syncytium formation and replication kinetics in vitro, although it is not the only factor implicated in HMPV disease severity in mice.

Virus-like particle vaccine induces cross-protection against human metapneumovirus infections in mice.

Human metapneumovirus (HMPV) is a paramyxovirus that causes acute respiratory-tract infections in children and adults worldwide. A safe and effective vaccine could decrease the burden of disease associated with this novel pathogen. We engineered HMPV viral-like particles (HMPV-VLPs) derived from retroviral core particles that mimic the properties of the viral surface of two HMPV viruses of either lineage A or B. These VLPs functionally display F and G HMPV surface glycoproteins. When injected in mice, HMPV-VLPs induce strong humoral immune response against both homologous and heterologous strains. Moreover, the induced neutralizing antibodies prevented mortality upon subsequent infection of the lungs with both homologous and heterologous viruses. Upon challenge, viral titers in the lungs of immunized animals were significantly reduced as compared to those of control animals. In conclusion, a HMPV-VLP vaccine that induces cross-protective immunity in mice is a promising approach to prevent HMPV infections.