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Basigin, or CD147, has been reported as a coreceptor used by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to invade host cells. Basigin also has a well-established role in Plasmodium falciparum malaria infection of human erythrocytes, where it is bound by one of the parasite’s invasion ligands, reticulocyte binding protein homolog 5 (RH5). Here, we sought to validate the claim that the receptor binding domain (RBD) of SARS-CoV-2 spike glycoprotein can form a complex with basigin, using RH5-basigin as a positive control. Using recombinantly expressed proteins, size exclusion chromatography and surface plasmon resonance, we show that neither RBD nor full-length spike glycoprotein bind to recombinant human basigin (expressed in either Escherichia coli or mammalian cells). Further, polyclonal anti-basigin IgG did not block SARS-CoV-2 infection of Vero E6 cells. Given the immense interest in SARS-CoV-2 therapeutic targets to improve treatment options for those who become seriously ill with coronavirus disease 2019 (COVID-19), we would caution the inclusion of basigin in this list on the basis of its reported direct interaction with SARS-CoV-2 spike glycoprotein. IMPORTANCE Reducing the mortality and morbidity associated with COVID-19 remains a global health priority. Vaccines have proven highly effective at preventing infection and hospitalization, but efforts must continue to improve treatment options for those who still become seriously ill. Critical to these efforts is the identification of host factors that are essential to viral entry and replication. Basigin, or CD147, was previously identified as a possible therapeutic target based on the observation that it may act as a coreceptor for SARS-CoV-2, binding to the receptor binding domain of the spike protein. Here, we show that there is no direct interaction between the RBD and basigin, casting doubt on its role as a coreceptor and plausibility as a therapeutic target.

Although recent epidemiological data suggest that pneumococci may contribute to the risk of SARS-CoV-2 disease, secondary pneumococcal pneumonia has been reported as infrequent. This apparent contradiction may be explained by interactions of SARS-CoV-2 and pneumococcus in the upper airway, resulting in the escape of SARS-CoV-2 from protective host immune responses. Here, we investigated the relationship of these two respiratory pathogens in two distinct cohorts of a) healthcare workers with asymptomatic or mildly symptomatic SARS-CoV-2 infection identified by systematic screening and b) patients with moderate to severe disease who presented to hospital. We assessed the effect of co-infection on host antibody, cellular and inflammatory responses to the virus. In both cohorts, pneumococcal colonisation was associated with diminished anti-viral immune responses, which affected primarily mucosal IgA levels among individuals with mild or asymptomatic infection and cellular memory responses in infected patients. Our findings suggest that S. pneumoniae modulates host immunity to SARS-CoV-2 and raises the question if pneumococcal carriage also enables immune escape of other respiratory viruses through a similar mechanism and facilitates reinfection occurrence.

Background COVID-19 vaccine supply shortages are causing concerns about compromised immunity in some countries as the interval between the first and second dose becomes longer. Conversely, countries with no supply constraints are considering administering a third dose. We assessed the persistence of immunogenicity after a single dose of ChAdOx1 nCoV-19 (AZD1222), immunity after an extended interval (44–45 weeks) between the first and second dose, and response to a third dose as a booster given 28–38 weeks after the second dose. Methods In this substudy, volunteers aged 18–55 years who were enrolled in the phase 1/2 (COV001) controlled trial in the UK and had received either a single dose or two doses of 5 × 1010 viral particles were invited back for vaccination. Here we report the reactogenicity and immunogenicity of a delayed second dose (44–45 weeks after first dose) or a third dose of the vaccine (28–38 weeks after second dose). Data from volunteers aged 18–55 years who were enrolled in either the phase 1/2 (COV001) or phase 2/3 (COV002), single-blinded, randomised controlled trials of ChAdOx1 nCoV-19 and who had previously received a single dose or two doses of 5 × 1010 viral particles are used for comparison purposes. COV001 is registered with ClinicalTrials.gov, NCT04324606, and ISRCTN, 15281137, and COV002 is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137, and both are continuing but not recruiting. Findings Between March 11 and 21, 2021, 90 participants were enrolled in the third-dose boost substudy, of whom 80 (89%) were assessable for reactogenicity, 75 (83%) were assessable for evaluation of antibodies, and 15 (17%) were assessable for T-cells responses. The two-dose cohort comprised 321 participants who had reactogenicity data (with prime-boost interval of 8–12 weeks: 267 [83%] of 321; 15–25 weeks: 24 [7%]; or 44–45 weeks: 30 [9%]) and 261 who had immunogenicity data (interval of 8–12 weeks: 115 [44%] of 261; 15–25 weeks: 116 [44%]; and 44–45 weeks: 30 [11%]). 480 participants from the single-dose cohort were assessable for immunogenicity up to 44–45 weeks after vaccination. Antibody titres after a single dose measured approximately 320 days after vaccination remained higher than the titres measured at baseline (geometric mean titre of 66·00 ELISA units [EUs; 95% CI 47·83–91·08] vs 1·75 EUs [1·60–1·93]). 32 participants received a late second dose of vaccine 44–45 weeks after the first dose, of whom 30 were included in immunogenicity and reactogenicity analyses. Antibody titres were higher 28 days after vaccination in those with a longer interval between first and second dose than for those with a short interval (median total IgG titre: 923 EUs [IQR 525–1764] with an 8–12 week interval; 1860 EUs [917–4934] with a 15–25 week interval; and 3738 EUs [1824–6625] with a 44–45 week interval). Among participants who received a third dose of vaccine, antibody titres (measured in 73 [81%] participants for whom samples were available) were significantly higher 28 days after a third dose (median total IgG titre: 3746 EUs [IQR 2047–6420]) than 28 days after a second dose (median 1792 EUs [IQR 899–4634]; Wilcoxon signed rank test p=0·0043). T-cell responses were also boosted after a third dose (median response increased from 200 spot forming units [SFUs] per million peripheral blood mononuclear cells [PBMCs; IQR 127–389] immediately before the third dose to 399 SFUs per milion PBMCs [314–662] by day 28 after the third dose; Wilcoxon signed rank test p=0·012). Reactogenicity after a late second dose or a third dose was lower than reactogenicity after a first dose. Interpretation An extended interval before the second dose of ChAdOx1 nCoV-19 leads to increased antibody titres. A third dose of ChAdOx1 nCoV-19 induces antibodies to a level that correlates with high efficacy after second dose and boosts T-cell responses. Funding UK Research and Innovation, Engineering and Physical Sciences Research Council, National Institute for Health Research, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research Oxford Biomedical Research Centre, Chinese Academy of Medical Sciences Innovation Fund for Medical Science, Thames Valley and South Midlands NIHR Clinical Research Network, AstraZeneca, and Wellcome.

There is an ongoing global effort to design, manufacture, and clinically assess vaccines against SARS-CoV-2. Over the course of the ongoing pandemic a number of new SARS-CoV-2 virus isolates or variants of concern (VoC) have been identified containing mutations in key proteins. In this study we describe the generation and preclinical assessment of a ChAdOx1-vectored vaccine (AZD2816) which expresses the spike protein of the Beta VoC (B.1.351). We demonstrate that AZD2816 is immunogenic after a single dose. When AZD2816 is used as a booster dose in animals primed with a vaccine encoding the original spike protein (ChAdOx1 nCoV-19/ [AZD1222]), high titre binding and neutralising antibodies against Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2) are induced. In addition, a strong and polyfunctional T cell response was measured in these booster regimens. These data support the ongoing clinical development and testing of this new variant vaccine.

Summary Reticulocyte-binding protein homolog 5 (RH5) is a leading Plasmodium falciparum blood-stage vaccine candidate. Another possible candidate, apical membrane antigen 1 (AMA1), was not efficacious in malaria-endemic populations, likely due to pre-existing antimalarial antibodies that interfered with the activity of vaccine-induced AMA1 antibodies, as judged by in vitro growth inhibition assay (GIA). To determine how pre-existing antibodies interact with vaccine-induced RH5 antibodies, we purify total and RH5-specific immunoglobulin Gs (IgGs) from malaria-exposed Malians and malaria-naive RH5 vaccinees. Infection-induced RH5 antibody titers are much lower than those induced by vaccination, and RH5-specific IgGs show differences in the binding site between the two populations. In GIA, Malian polyclonal IgGs show additive or synergistic interactions with RH5 human monoclonal antibodies and overall additive interactions with vaccine-induced polyclonal RH5 IgGs. These results suggest that pre-existing antibodies will interact favorably with vaccine-induced RH5 antibodies, in contrast to AMA1 antibodies. This study supports RH5 vaccine trials in malaria-endemic regions., Graphical abstract, Highlights RH5 IgG titers induced by infection are lower than those induced by RH5 vaccination Infection- and vaccine-induced RH5 IgGs have different specificity and avidity Infection- and vaccine-induced RH5 IgGs interact differently with RH5 mAbs Infection-induced IgGs generally do not reduce the activity of vaccine-induced IgGs, Willcox et al. combine antibodies from malaria-exposed Malians and volunteers who received a reticulocyte-binding protein homolog 5 (RH5) vaccine. In P. falciparum growth inhibition assays, infection-induced IgGs generally do not reduce the neutralizing activity of vaccine-induced IgGs. These results suggest that RH5 vaccines will induce effective antibodies in malaria-endemic populations.

In order to assess the anthropogenic impacts on groundwater quality we generally need to identify the natural conditions or Natural background levels (NBLs) within groundwater systems, which are used as references to assess the evolution of the contamination status. This information, in addition to the threshold values (TVs), which are derived from NBLs and based on specific criteria values for legitimate water uses and the environment (terrestrial and aquatic ecosystems), is required to appropriately assess of the chemical status of groundwater in accordance with the European Water Framework Directive and to analyse and propose potential measures where they are required.In literature, different methodologies are available to assess NBLs and reference TVs for different geochemical substances based on the available measurements of groundwater salinity (e.g. based on probability plots). In this work we analyze and compare three previously proposed methods for derivation of chloride NBLs, which is a conservation solute and is closely related to salt water intrusion in inland and coastal aquifers. We discuss and compare their applicability in pilots located in different settings (southern /northern European / Mediteranean/ North Sea /Baltic Sea) covering different typologies (detrital, karstic, fissured aquifers) and management issues (overexploitation, land use and land cover changes, etc). We perform sensitivity analysis to different constraints applied to remove samples affected by human activity (Nitrate and the brackish saline constraints) in the assessment of NBLs. Finally, based on this analysis, we propose a general approach for derivation of NBLs that could be applied to any of the tested pilots as well as other similar settings in Europe. This research has been partially supported by the SIGLO-AN project (RTI2018-101397-B-I00) from the Spanish Ministry of Science, Innovation and Universities (Programa Estatal de I+D+I orientada a los Retos de la Sociedad).This work has been partially supported by the GeoE.171.008-TACTIC and GeoE.171.008-HOVER projects from GeoERA organization funded by European Union’s Horizon 2020 research and innovation program.

Basigin, or CD147, has been reported as a co-receptor used by SARS-CoV-2 to invade host cells. Basigin also has a well-established role in Plasmodium falciparum malaria infection of human erythrocytes where it is bound by one of the parasite’s invasion ligands, reticulocyte binding protein homolog 5 (RH5). Here, we sought to validate the claim that the receptor binding domain (RBD) of SARS-CoV-2 spike glycoprotein can form a complex with basigin, using RH5-basigin as a positive control. Using recombinantly expressed proteins, size exclusion chromatography and surface plasmon resonance, we show that neither RBD nor full-length spike glycoprotein bind to recombinant human basigin (either expressed in E. coli or mammalian cells). Given the immense interest in SARS-CoV-2 therapeutic targets, we would caution the inclusion of basigin in this list on the basis of its reported direct interaction with SARS-CoV-2 spike glycoprotein.ImportanceReducing the mortality and morbidity associated with COVID-19 remains a global health priority. Critical to these efforts is the identification of host factors that are essential to viral entry and replication. Basigin, or CD147, was previously identified as a possible therapeutic target based on the observation that it may act as a co-receptor for SARS-COV-2, binding to the receptor binding domain of the spike protein. Here, we show that there is no direct interaction between the RBD and basigin, casting doubt on its role as a co-receptor and plausibility as a therapeutic target.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. \ud \ud \ud METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. \ud \ud \ud FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. \ud \ud \ud INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. \ud \ud \ud FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca.

BACKGROUND: Older adults (aged ≥70 years) are at increased risk of severe disease and death if they develop COVID-19 and are therefore a priority for immunisation should an efficacious vaccine be developed. Immunogenicity of vaccines is often worse in older adults as a result of immunosenescence. We have reported the immunogenicity of a novel chimpanzee adenovirus-vectored vaccine, ChAdOx1 nCoV-19, in young adults, and now describe the safety and immunogenicity of this vaccine in a wider range of participants, including adults aged 70 years and older. METHODS: In this report of the phase 2 component of a single-blind, randomised, controlled, phase 2/3 trial (COV002), healthy adults aged 18 years and older were enrolled at two UK clinical research facilities, in an age-escalation manner, into 18-55 years, 56-69 years, and 70 years and older immunogenicity subgroups. Participants were eligible if they did not have severe or uncontrolled medical comorbidities or a high frailty score (if aged ≥65 years). First, participants were recruited to a low-dose cohort, and within each age group, participants were randomly assigned to receive either intramuscular ChAdOx1 nCoV-19 (2·2 × 1010 virus particles) or a control vaccine, MenACWY, using block randomisation and stratified by age and dose group and study site, using the following ratios: in the 18-55 years group, 1:1 to either two doses of ChAdOx1 nCoV-19 or two doses of MenACWY; in the 56-69 years group, 3:1:3:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY; and in the 70 years and older, 5:1:5:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY. Prime-booster regimens were given 28 days apart. Participants were then recruited to the standard-dose cohort (3·5-6·5 × 1010 virus particles of ChAdOx1 nCoV-19) and the same randomisation procedures were followed, except the 18-55 years group was assigned in a 5:1 ratio to two doses of ChAdOx1 nCoV-19 or two doses of MenACWY. Participants and investigators, but not staff administering the vaccine, were masked to vaccine allocation. The specific objectives of this report were to assess the safety and humoral and cellular immunogenicity of a single-dose and two-dose schedule in adults older than 55 years. Humoral responses at baseline and after each vaccination until 1 year after the booster were assessed using an in-house standardised ELISA, a multiplex immunoassay, and a live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) microneutralisation assay (MNA80). Cellular responses were assessed using an ex-vivo IFN-γ enzyme-linked immunospot assay. The coprimary outcomes of the trial were efficacy, as measured by the number of cases of symptomatic, virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were by group allocation in participants who received the vaccine. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. This study is ongoing and is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137. FINDINGS: Between May 30 and Aug 8, 2020, 560 participants were enrolled: 160 aged 18-55 years (100 assigned to ChAdOx1 nCoV-19, 60 assigned to MenACWY), 160 aged 56-69 years (120 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY), and 240 aged 70 years and older (200 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY). Seven participants did not receive the boost dose of their assigned two-dose regimen, one participant received the incorrect vaccine, and three were excluded from immunogenicity analyses due to incorrectly labelled samples. 280 (50%) of 552 analysable participants were female. Local and systemic reactions were more common in participants given ChAdOx1 nCoV-19 than in those given the control vaccine, and similar in nature to those previously reported (injection-site pain, feeling feverish, muscle ache, headache), but were less common in older adults (aged ≥56 years) than younger adults. In those receiving two standard doses of ChAdOx1 nCoV-19, after the prime vaccination local reactions were reported in 43 (88%) of 49 participants in the 18-55 years group, 22 (73%) of 30 in the 56-69 years group, and 30 (61%) of 49 in the 70 years and older group, and systemic reactions in 42 (86%) participants in the 18-55 years group, 23 (77%) in the 56-69 years group, and 32 (65%) in the 70 years and older group. As of Oct 26, 2020, 13 serious adverse events occurred during the study period, none of which were considered to be related to either study vaccine. In participants who received two doses of vaccine, median anti-spike SARS-CoV-2 IgG responses 28 days after the boost dose were similar across the three age cohorts (standard-dose groups: 18-55 years, 20 713 arbitrary units [AU]/mL [IQR 13 898-33 550], n=39; 56-69 years, 16 170 AU/mL [10 233-40 353], n=26; and ≥70 years 17 561 AU/mL [9705-37 796], n=47; p=0·68). Neutralising antibody titres after a boost dose were similar across all age groups (median MNA80 at day 42 in the standard-dose groups: 18-55 years, 193 [IQR 113-238], n=39; 56-69 years, 144 [119-347], n=20; and ≥70 years, 161 [73-323], n=47; p=0·40). By 14 days after the boost dose, 208 (>99%) of 209 boosted participants had neutralising antibody responses. T-cell responses peaked at day 14 after a single standard dose of ChAdOx1 nCoV-19 (18-55 years: median 1187 spot-forming cells [SFCs] per million peripheral blood mononuclear cells [IQR 841-2428], n=24; 56-69 years: 797 SFCs [383-1817], n=29; and ≥70 years: 977 SFCs [458-1914], n=48). INTERPRETATION: ChAdOx1 nCoV-19 appears to be better tolerated in older adults than in younger adults and has similar immunogenicity across all age groups after a boost dose. Further assessment of the efficacy of this vaccine is warranted in all age groups and individuals with comorbidities. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midlands NIHR Clinical Research Network, and AstraZeneca.

Background: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2. Methods: We did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18–55 years with no history of laboratory confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5 × 10 10 viral particles or MenACWY as a single intramuscular injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque reduction neutralisation assay [PRNT 50]; a microneutralisation assay [MNA 50, MNA 80, and MNA 90]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virologically confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606. Findings: Between April 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p80 and in 35 (100%) participants when measured in PRNT 50. After a booster dose, all participants had neutralising activity (nine of nine in MNA 80 at day 42 and ten of ten in Marburg VN on day 56). Neutralising antibody responses correlated strongly with antibody levels measured by ELISA (R 2=0·67 by Marburg VN; p

Eradication of established biofilm communities of pathogenic bacteria is one of the pending challenges in the development of new antimicrobial agents. In particular, the dreaded nosocomial Pseudomonas aeruginosa forms microbial communities that offer an enhanced resistance to conventional antibiotics. Recently, we have described an engineered antimicrobial peptide derived from the human RNase3, also named the eosinophil cationic protein (ECP), RN3 (5–36), which combines bactericidal activity with high cell agglutination and lipopolysaccharide (LPS) affinity. Through a single replacement scan library using the SPOT methodology we have evaluated both the contribution of sequence positioning and amino acid singularity towards the peptide biological and physicochemical properties. Results indicate that the ECP N-terminus has already been extensively improved through evolution to provide high antimicrobial activity; hence most substitutions improving its antimicrobial performance are in detriment of safety towards host tissues. Only three positions were identified, occupied by polar residues on the first α-helix of the protein and replaceable by a hydrophobic residue, allowing an extended N-terminal patch that mediates bacterial agglutination. Among the best candidates, an Ile replacement proved best in improving the peptide therapeutic window. The novel engineered peptides encompass both the LPS-binding and aggregation-prone regions of parental ECP, providing the appropriate structural features for peptide attachment to the bacterial exopolysaccharide layer and bacterial cell membrane destabilization, thereby promoting biofilm removal at micro molar concentrations. We conclude that the novel engineered peptides are promising lead candidates against Gram-negative biofilms.

Summary Background Development of an effective vaccine against the pathogenic blood-stage infection of human malaria has proved challenging, and no candidate vaccine has affected blood-stage parasitemia following controlled human malaria infection (CHMI) with blood-stage Plasmodium falciparum. Methods We undertook a phase I/IIa clinical trial in healthy adults in the United Kingdom of the RH5.1 recombinant protein vaccine, targeting the P. falciparum reticulocyte-binding protein homolog 5 (RH5), formulated in AS01B adjuvant. We assessed safety, immunogenicity, and efficacy against blood-stage CHMI. Trial registered at ClinicalTrials.gov, NCT02927145. Findings The RH5.1/AS01B formulation was administered using a range of RH5.1 protein vaccine doses (2, 10, and 50 μg) and was found to be safe and well tolerated. A regimen using a delayed and fractional third dose, in contrast to three doses given at monthly intervals, led to significantly improved antibody response longevity over ∼2 years of follow-up. Following primary and secondary CHMI of vaccinees with blood-stage P. falciparum, a significant reduction in parasite growth rate was observed, defining a milestone for the blood-stage malaria vaccine field. We show that growth inhibition activity measured in vitro using purified immunoglobulin G (IgG) antibody strongly correlates with in vivo reduction of the parasite growth rate and also identify other antibody feature sets by systems serology, including the plasma anti-RH5 IgA1 response, that are associated with challenge outcome. Conclusions Our data provide a new framework to guide rational design and delivery of next-generation vaccines to protect against malaria disease. Funding This study was supported by USAID, UK MRC, Wellcome Trust, NIAID, and the NIHR Oxford-BRC., Graphical abstract, Highlights The RH5.1/AS01B vaccine is safe, well tolerated, and immunogenic in healthy adults A delayed fractional third dose significantly improves antibody response longevity In vivo blood-stage P. falciparum growth rate is significantly lower in vaccinees In vitro IgG-mediated growth inhibition activity is associated with challenge outcome, Context and significance A highly effective vaccine against the human malaria parasite Plasmodium falciparum is urgently needed. One vaccine strategy aims to prevent parasite growth in the blood, protecting against clinical disease; however, this has proved exceptionally challenging. Here we show that a candidate vaccine (reticulocyte-binding protein homolog 5.1 [RH5.1]/AS01B) is safe in a phase I/IIa clinical trial and identify a vaccination regimen that improves the durability of the human antibody response, which is critical for long-term protection. Following experimental challenge of vaccinated adults with malaria, we observed that the vaccine could reduce parasite growth in the blood and identified immune responses that could predict how well the vaccine performs. These data will help guide the design of improved vaccines in the future., Minassian et al. report that the RH5.1/AS01B vaccine against blood-stage Plasmodium falciparum malaria is safe and immunogenic in a phase I/IIa clinical trial. They demonstrate a significantly reduced blood-stage parasite growth rate in vaccinees following controlled human malaria infection and identify that in vitro antibody-mediated growth inhibition activity is associated with challenge outcome.

Laminins are a major constituent of all basement membranes. The polymerisation of laminins at the cell surface is mediated by the three short arms of the cross-shaped laminin heterotrimer. The short arms contain repeats of laminin-type epidermal growth factor-like (LE) domains, interspersed with globular domains of unknown function. A single LF domain is inserted between LE5 and LE6 of the laminin β1 and β2 chains. We report the crystal structure at 1.85 Å resolution of the laminin β2 LE5-LF-LE6 region. The LF domain consists of a β-sandwich related to bacterial family 35 carbohydrate binding modules, and more distantly to the L4 domains present in the short arms of laminin α and γ chains. An α-helical region mediates the extensive interaction of the LF domain with LE5. The relative arrangement of LE5 and LE6 is very similar to that of consecutive LE domains in uninterrupted LE tandems. Fitting atomic models to a low-resolution structure of the first eight domains of the laminin β1 chain determined by small-angle X-ray scattering suggests a deviation from the regular LE array at the LE4–LE5 junction. These results advance our understanding of laminin structure., Highlights • The crystal structure of the laminin β2 LE5-LF-L6 region has been determined. • The LF domain is a β-sandwich with distant homology to the L4 domains in laminin α and γ chains. • A unique α-helical region in the LF domain interacts extensively with LE5. • LE5 and LE6 are arranged in a manner typical of tandem LE domains, despite the insertion of the LF domain.

Candida albicans is a polymorphic fungus responsible for mucosal and skin infections. Candida cells establish themselves into biofilm communities resistant to most currently available antifungal agents. An increase of severe infections ensuing in fungal septic shock in elderly or immunosuppressed patients, along with the emergence of drug-resistant strains, urge the need for the development of alternative antifungal agents. In the search for novel antifungal drugs our laboratory demonstrated that two human ribonucleases from the vertebrate-specific RNaseA superfamily, hRNase3 and hRNase7, display a high anticandidal activity. In a previous work, we proved that the N-terminal region of the RNases was sufficient to reproduce most of the parental protein bactericidal activity. Next, we explored their potency against a fungal pathogen. Here, we have tested the N-terminal derived peptides that correspond to the eight human canonical RNases (RN1-8) against planktonic cells and biofilms of C. albicans. RN3 and RN7 peptides displayed the most potent inhibitory effect with a mechanism of action characterized by cell-wall binding, membrane permeabilization and biofilm eradication activities. Both peptides are able to eradicate planktonic and sessile cells, and to alter their gene expression, reinforcing its role as a lead candidate to develop novel antifungal and antibiofilm therapies. This research was funded by the Ministerio de Economía y Competitividad (SAF2017-82158-R and SAF2015-66007P, Fundació La Marató de TV3 (20180310) and Generalitat de Catalunya (2016-PROD-00060). M.T. would like to acknowledge support from the Programa Ramon y Cajal (RYC-2012-09999) and the European Society of Clinical Microbiology and Infectious Diseases though ans ESCMID-2016 grant.

Stroke affects the population worldwide, with a prevalence of 0.58% worldwide. One of the possible consequences is the negative impact in the motor function of the patient, limiting their quality of life. For these reason, Brain-Computer Interfaces are studied as a tool for improving rehabilitation processes. Nevertheless, to the best of our knowledge, there are no Brain-Computer Interface systems which use video-games for upper limb motor rehabilitation. This study aimed to design and assess a Human-Computer Interface that includes electroencephalography, forearm motion and postural analysis, with healthy subjects. This assessment was made by designing two scenarios in which the participant carried out exercises involving the mouth and the hand and forearm trajectory symmetry. Results show that the system is ready to be tested on patients, since the participants were comfortable using it. Also, the quantitative results, particularly, the metrics used in the video-game, are an important start for health professionals to characterize motor rehabilitation in stroke patients, enabling the path to the use of the designed system in motor rehabilitation therapies.

The development of novel treatment against tuberculosis is a priority global health challenge. Antimicrobial proteins and peptides offer a multifaceted mechanism suitable to fight bacterial resistance. Within the RNaseA superfamily there is a group of highly cationic proteins secreted by innate immune cells with anti-infective and immune-regulatory properties. In this work, we have tested the human canonical members of the RNase family using a spot-culture growth inhibition assay based mycobacteria-infected macrophage model for evaluating their anti-tubercular properties. Out of the seven tested recombinant human RNases, we have identified two members, RNase3 and RNase6, which were highly effective against Mycobacterium aurum extra- and intracellularly and induced an autophagy process. We observed the proteins internalization within macrophages and their capacity to eradicate the intracellular mycobacterial infection at a low micro-molar range. Contribution of the enzymatic activity was discarded by site-directed mutagenesis at the RNase catalytic site. The protein induction of autophagy was analyzed by RT-qPCR, western blot, immunofluorescence, and electron microscopy. Specific blockage of auto-phagosome formation and maturation reduced the protein's ability to eradicate the infection. In addition, we found that the M. aurum infection of human THP1 macrophages modulates the expression of endogenous RNase3 and RNase6, suggesting a function in vivo. Overall, our data anticipate a biological role for human antimicrobial RNases in host response to mycobacterial infections and set the basis for the design of novel anti-tubercular drugs.

Eradication of established biofilm communities of pathogenic Gram-negative species is one of the pending challenges for the development of new antimicrobial agents. In particular, Pseudomonas aeruginosa is one of the main dreaded nosocomial species, with a tendency to form organized microbial communities that offer an enhanced resistance to conventional antibiotics. We describe here an engineered antimicrobial peptide (AMP) which combines bactericidal activity with a high bacterial cell agglutination and lipopolysaccharide (LPS) affinity. The RN3(5-17P22-36) peptide is a 30-mer derived from the eosinophil cationic protein (ECP), a host defense RNase secreted by eosinophils upon infection, with a wide spectrum of antipathogen activity. The protein displays high biofilm eradication activity that is not dependent on its RNase catalytic activity, as evaluated by using an active site-defective mutant. On the other hand, the peptide encompasses both the LPS-binding and aggregation-prone regions from the parental protein, which provide the appropriate structural features for the peptide's attachment to the bacterial exopolysaccharide layer and further improved removal of established biofilms. Moreover, the peptide's high cationicity and amphipathicity promote the cell membrane destabilization action. The results are also compared side by side with other reported AMPs effective against either planktonic and/or biofilm forms of Pseudomonas aeruginosa strain PAO1. The ECP and its derived peptide are unique in combining high bactericidal potency and cell agglutination activity, achieving effective biofilm eradication at a low micromolar range. We conclude that the designed RN3(5-17P22-36) peptide is a promising lead candidate against Gram-negative biofilms.

Lipopolysaccharides (LPSs) and glycosaminoglycans (GAGs) are polymeric structures containing negatively charged disaccharide units that bind to specialized proteins and peptides in the human body and control fundamental processes such as inflammation and coagulation. Surprisingly, some proteins can bind both LPSs and GAGs with high affinity, suggesting that a cross-communication between these two pathways can occur. Here, we explore whether GAGs and LPSs can share common binding sites in proteins and what are the structural determinants of this binding. We found that the LPS-binding peptide YI12WF, derived from protein FhuA, can bind both heparin andE. coliLPS with high affinity. Most interestingly, mutations decreasing heparin binding in the peptide also reduce LPS affinity. We show that such mutations involve the CPC clip motif in the peptide, a small three-dimensional signature required for heparin binding. Overall, we conclude that negatively charged polysaccharide-containing polymers such as GAGs and LPSs can compete for similar binding sites in proteins, and that the CPC clip motif is essential to bind both ligands. Our results provide a structural framework to explain why these polymers can cross-interact with the same proteins and peptides and thus contribute to the regulation of apparently unrelated processes in the body.

Tuberculosis (TB) continues to be a devastating infectious disease and remerges as a global health emergency due to an alarming rise of antimicrobial resistance to its treatment. Despite of the serious effort that has been applied to develop effective antitubercular chemotherapies, the potential of antimicrobial peptides (AMPs) remains underexploited. A large amount of literature is now accessible on the AMP mechanisms of action against a diversity of pathogens; nevertheless, research on their activity on mycobacteria is still scarce. In particular, there is an urgent need to integrate all available interdisciplinary strategies to eradicate extensively drug-resistant Mycobacterium tuberculosis strains. In this context, we should not underestimate our endogenous antimicrobial proteins and peptides as ancient players of the human host defense system. We are confident that novel antibiotics based on human AMPs displaying a rapid and multifaceted mechanism, with reduced toxicity, should significantly contribute to reverse the tide of antimycobacterial drug resistance. In this review, we have provided an up to date perspective of the current research on AMPs to be applied in the fight against TB. A better understanding on the mechanisms of action of human endogenous peptides should ensure the basis for the best guided design of novel antitubercular chemotherapeutics.

Knowledge on the contribution of protein glycosylation in host defense antimicrobial peptides is still scarce. We have studied here how the post-translational modification pattern modulates the antimicrobial activity of one of the best characterized leukocyte granule proteins. The human eosinophil cationic protein (ECP), an eosinophil specific granule protein secreted during inflammation and infection, can target a wide variety of pathogens. Previous work in human eosinophil extracts identified several ECP native forms and glycosylation heterogeneity was found to contribute to the protein biological properties. In this study we analyze for the first time the antimicrobial activity of the distinct native proteins purified from healthy donor blood. Low and heavy molecular weight forms were tested on Escherichia coli cell cultures and compared with the recombinant non-glycosylated protein. Further analysis on model membranes provided an insight towards an understanding of the protein behavior at the cytoplasmic membrane level. The results highlight the significant reduction in protein toxicity and bacteria agglutination activity for heavy glycosylated fractions. Notwithstanding, the lower glycosylated fraction mostly retains the lipopolysaccharide binding affinity together with the cytoplasmic membrane depolarization and membrane leakage activities. From structural analysis we propose that heavy glycosylation interferes with the protein self-aggregation, hindering the cell agglutination and membrane disruption processes. The results suggest the contribution of post-translational modifications to the antimicrobial role of ECP in host defense.

Human Ribonuclease 6 is a secreted protein belonging to the ribonuclease A (RNaseA) superfamily, a vertebrate specific family suggested to arise with an ancestral host defense role. Tissue distribution analysis revealed its expression in innate cell types, showing abundance in monocytes and neutrophils. Recent evidence of induction of the protein expression by bacterial infection suggested an antipathogen function in vivo. In our laboratory, the antimicrobial properties of the protein have been evaluated against Gram-negative and Gram-positive species and its mechanism of action was characterized using a membrane model. Interestingly, our results indicate that RNase6, as previously reported for RNase3, is able to specifically agglutinate Gram-negative bacteria as a main trait of its antimicrobial activity. Moreover, a side by side comparative analysis with the RN6(1-45) derived peptide highlights that the antimicrobial activity is mostly retained at the protein N-terminus. Further work by site directed mutagenesis and structural analysis has identified two residues involved in the protein antimicrobial action (Trp1 and Ile13) that are essential for the cell agglutination properties. This is the first structure-functional characterization of RNase6 antimicrobial properties, supporting its contribution to the infection focus clearance.

Diseases caused by protozoan parasites can pose a severe thread to human health and are behind some serious neglected tropical diseases like malaria and leishmaniasis. Though several different drugs have been developed in order to eradicate these diseases, a successful candidate has not yet been discovered. Among the most active compounds tested, antimicrobial peptides (AMPs) are particularly appealing because of their wide spectrum of action. AMPs have been described to perturb protozoan homeostasis by disrupting the cellular membranes but also by interfering with key processes in the parasite metabolism. In this review we describe the diverse mechanisms of action of AMPs on protozoan targets and how they can be exploited to treat diseases. Moreover, we describe with detail the antimicrobial action of AMPs on two major parasitical infections: leishmaniasis and malaria. All the features reviewed here show that AMPs are promising drugs to target protozoan parasites and that further understanding of the mechanism of action of these compounds will lead to improved drugs that could be worth to test in a clinical phase.

Antimicrobial RNases are small cationic proteins belonging to the vertebrate RNase A superfamily and endowed with a wide range of antipathogen activities. Vertebrate RNases, while sharing the active site architecture, are found to display a variety of noncatalytical biological properties, providing an excellent example of multitask proteins. The antibacterial activity of distant related RNases suggested that the family evolved from an ancestral host-defence function. The review provides a structural insight into antimicrobial RNases, taking as a reference the human RNase 3, also named eosinophil cationic protein (ECP). A particular high binding affinity against bacterial wall structures mediates the protein action. In particular, the interaction with the lipopolysaccharides at the Gram-negative outer membrane correlates with the protein antimicrobial and specific cell agglutinating activity. Although a direct mechanical action at the bacteria wall seems to be sufficient to trigger bacterial death, a potential intracellular target cannot be discarded. Indeed, the cationic clusters at the protein surface may serve both to interact with nucleic acids and cell surface heterosaccharides. Sequence determinants for ECP activity were screened by prediction tools, proteolysis and peptide synthesis. Docking results are complementing the structural analysis to delineate the protein anchoring sites for anionic targets of biological significance.

Antimicrobial proteins and peptides (AMPs) are essential effectors of innate immunity, acting as a first line of defense against bacterial infections. Many AMPs exhibit high affinity for cell wall structures such as lipopolysaccharide (LPS), a potent endotoxin able to induce sepsis. Hence, understanding how AMPs can interact with and neutralize LPS endotoxin is of special relevance for human health. Eosinophil cationic protein (ECP) is an eosinophil secreted protein with high activity against both Gram-negative and Gram-positive bacteria. ECP has a remarkable affinity for LPS and a distinctive agglutinating activity. By using a battery of LPS-truncated E. coli mutant strains, we demonstrate that the polysaccharide moiety of LPS is essential for ECP-mediated bacterial agglutination, thereby modulating its antimicrobial action. The mechanism of action of ECP at the bacterial surface is drastically affected by the LPS structure and in particular by its polysaccharide moiety. We have also analyzed an N-terminal fragment that retains the whole protein activity and displays similar cell agglutination behavior. Conversely, a fragment with further minimization of the antimicrobial domain, though retaining the antimicrobial capacity, significantly loses its agglutinating activity, exhibiting a different mechanism of action which is not dependent on the LPS composition. The results highlight the correlation between the protein's antimicrobial activity and its ability to interact with the LPS outer layer and promote bacterial agglutination.

Study region This study is focused on 12 basins in mainland Spain where monthly series longer than 30 years of near-natural streamflow measurements are available. It covers areas with different climate conditions. Study focus The potential impact of future climate change scenarios on water resources in the Spanish basins is studied. It takes into account uncertainties in the estimation of local climate conditions, and the propagation of the impact due to the structural uncertainties related with the adopted conceptual-numerical approach. Local climate scenarios are derived from available Regional Climate Model (RCM) simulations after statistical downscaling. The future scenarios have been generated assuming two hypotheses of future warming for two basins in mainland Spain: 1.5 oC and 3 oC. In each of these basins, the local climate scenarios have been propagated by using 4 hydrological models, with sufficient capacity to reproduce the historical dynamic, providing values of above 5 in a 0–9 range for the grading method used. New hydrological insights The results show a significant spatial heterogeneity of the impact of climate change on the mean streamflow in Spanish basins. The highest reductions of flow appear in the wetter northern basins. The seasonality of the impact is also significant, with the highest reductions during autumn, and the smallest changes in the summer months. Finally, the highest uncertainties in this climate change impact assessment are due to the RCM projections, with the influence of the hydrological models being significantly smaller.