Data_Sheet_1_Antibiotic resistance gene sequencing is necessary to reveal the complex dynamics of immigration from sewers to activated sludge.docx

Microbial community composition has increasingly emerged as a key determinant of antibiotic resistance gene (ARG) content. However, in activated sludge wastewater treatment plants (AS-WWTPs), a comprehensive understanding of the microbial community assembly process and its impact on the persistence of antimicrobial resistance (AMR) remains elusive. An important part of this process is the immigration dynamics (or community coalescence) between the influent and activated sludge. While the influent wastewater contains a plethora of ARGs, the persistence of a given ARG depends initially on the immigration success of the carrying population, and the possible horizontal transfer to indigenously resident populations of the WWTP. The current study utilized controlled manipulative experiments that decoupled the influent wastewater composition from the influent microbial populations to reveal the fundamental mechanisms involved in ARG immigration between sewers and AS-WWTP. A novel multiplexed amplicon sequencing approach was used to track different ARG sequence variants across the immigration interface, and droplet digital PCR was used to quantify the impact of immigration on the abundance of the targeted ARGs. Immigration caused an increase in the abundance of over 70 % of the quantified ARGs. However, monitoring of ARG amplicon sequence variants (ARG-ASVs) at the immigration interface revealed various immigration patterns such as (i) suppression of the indigenous mixed liquor ARG-ASV by the immigrant, or conversely (ii) complete immigration failure of the influent ARG-ASV. These immigration profiles are reported for the first time here and highlight the crucial information that can be gained using our novel multiplex amplicon sequencing techniques. Future studies aiming to reduce AMR in WWTPs should consider the impact of influent immigration in process optimisation and design.</p

Identification of Pre-Erythrocytic Malaria Antigens That Target Hepatocytes for Killing <i>In Vivo</i> and Contribute to Protection Elicited by Whole-Parasite Vaccination

<div><p>Pre-erythrocytic malaria vaccines, including those based on whole-parasite approaches, have shown protective efficacy in animal and human studies. However few pre-erythocytic antigens other than the immunodominant circumsporozoite protein (CSP) have been studied in depth with the goal of developing potent subunit malaria vaccines that are suited for use in endemic areas. Here we describe a novel technique to identify pre-erythrocytic malaria antigens that contribute to protection elicited by whole-parasite vaccination in the mouse model. Our approach combines immunization with genetically attenuated parasites and challenge with DNA plasmids encoding for potential protective pre-erythrocytic malaria antigens as luciferase fusions by hydrodynamic tail vein injection. After optimizing the technique, we first showed that immunization with <i>Pyfabb/f⁻</i>, a <i>P. yoelii</i> genetically attenuated parasite, induces killing of CSP-presenting hepatocytes. Depletion of CD8⁺ but not CD4⁺ T cells diminished the killing of CSP-expressing hepatocytes, indicating that killing is CD8⁺ T cell-dependent. Finally we showed that the use of heterologous prime/boost immunization strategies that use genetically attenuated parasites and DNA vaccines enabled the characterization of a novel pre-erythrocytic antigen, Tmp21, as a contributor to <i>Pyfabb/f⁻</i> induced protection. This technique will be valuable for identification of potentially protective liver stage antigens and has the potential to contribute to the understanding of immunity elicited by whole parasite vaccination, as well as the development of effective subunit malaria vaccines.</p></div

<i>Py</i>CSP-specific CD8⁺ T cells produce high levels of IFN-γ.

<p>(A) Characterization of infiltrating liver lymphocytes isolated from mice immunized with <i>Pyfabb/f</i>⁻ sporozoites (bottom panel) or mock-immunized (top panel) 7 days after HTVI challenge with plasmid DNA encoding for <i>Py</i>CSP-Luc. Representative dot plots gated on live lymphocytes showing CD8⁺ versus CD3⁺ (left panel) and CD8⁺ versus H-2Kd CSP tetramer (left panel) expression (B) Absolute number of CD8⁺ T cells in mock-immunized mice and in mice immunized with <i>Pyfabb/f</i>⁻ sporozoites. (C) Percentage of CSP-specific CD8⁺ T cells in mock-immunized mice and in mice immunized with <i>Pyfabb/f</i>⁻ sporozoites. (D) Characterization of IFN-γ producing liver CD8⁺ T cells in mice immunized with <i>Pyfabb/f</i>⁻ sporozoites (bottom panel) or mock-immunized (top panel) as determined by intracellular cytokine staining. Representative dot plots gated on live CD3⁺ lymphocytes showing CD8⁺ versus IFN-γ expression. (E) Representative dot plot gated on live CD3⁺ CD8⁺ T cells showing H-2Kd-CSP versus IFN-γ expression. (F) Absolute number of IFN-γ⁺ CD8⁺ T cells in in mock-immunized mice and in immunized with <i>Pyfabb/f</i>⁻ sporozoites. (G) Percentage of CSP-specific CD8⁺ T cells producing IFN-γ in mock-immunized mice and in mice immunized with <i>Pyfabb/f</i>⁻ sporozoites. (B–C and F–G) The data represents 3–5 individually analyzed mice in each group, and correspond to mean ± SD; significant differences between the mean of the mock vs. <i>Pyfabb/f</i>⁻-immunized mice calculated using the Mann-Whitney test are indicated (* = <i>p</i><0.05).</p

DNA vaccination of mice with <i>Py</i>Tmp21 reduces liver stage parasite burden.

<p>Shown is the average ratio of <i>P. yoelii</i> 18S RNA to GAPDH mouse housekeeping gene mRNA for 3–5 individually analyzed mice immunized with gWIZ, <i>Py</i>CSP or <i>Py</i>Tmp21 plasmid DNA. Statistical significance between the <i>Py</i>CSP or <i>Py</i>Tmp21 as compared to gWIZ plasmid was calculated using the Mann-Whitney test (* = <i>p</i><0.05).</p

Immunization of mice with <i>Pyfabb/f⁻</i> reduces <i>Py</i>CSP-Luc <i>in vivo</i> luminescence.

<p>(A–B) Expression of luciferase (Luc) and <i>Py</i>CSP-Luc in the liver of mice immunized by HTVI. (A) Luciferase signal in live naïve BALB/c mice injected by HTVI with 25 µg of either phCMV-Luc plasmid DNA (top panel) or phCMV-<i>Py</i>CSP-Luc (bottom panel) and imaged 8 h later by IVIS, after injection of D-luciferin. The scale indicates radiance expressed as p/s/cm²/sr. (B) Kinetics of luciferase signal in live naïve BALB/c mice (shown as total flux p per second) during the course of the experiment. Data is representative of two individual experiments, with 3 mice each per group. (C–E) Reduction of luciferase signal in the liver of <i>Pyfabb/f⁻</i>-immunized mice upon HTVI challenge with <i>Py</i>CSP-Luc plasmid DNA (C) Luciferase signal in 3 representative live mice immunized twice with 50,000 <i>Pyfabb/f⁻</i> salivary gland sporozoites (right panel) or mock-immunized with salivary gland debris (left panel), challenged by HTVI with 25 µg of phCMV-Luc plasmid DNA (top panel) <i>Py</i>CSP-Luc (bottom panel) 30 days after the last immunization, imaged 7 d post challenge as described. Each group contained 4 to 5 mice. (D) Quantification of luciferase signal (shown as total flux p per second) from mice in the four groups described in part C. The data represents 4 to 5 individually analyzed mice in each group and correspond to mean ± SD; significant differences between the mean of the mock vs. <i>Pyfabb/f⁻</i>-immunized mice for each plasmid calculated using the Mann-Whitney test are indicated (** = <i>p</i><0.01). (E) Inhibition of luciferase signal over the course of the experiment for mice challenged with plasmid DNA encoding for luciferase alone (Luc, red triangles) or <i>Py</i>CSP-Luc (blue squares), calculated as percentage reduction vs. the mock-immunization control). The data represents 4 to 5 individually analyzed mice in each group and correspond to mean ± SD; significant differences between the mean of Luc vs. <i>Py</i>CSP-Lus calculated using the Mann-Whitney test are indicated for each plasmid (* = <i>p</i><0.005; ** = <i>p</i><0.001).</p

<i>Py</i>Tmp21 contributes to the protective immune response elicited by GAP vaccines.

<p>(A) Quantification of luciferase signal (shown as total flux p per second) in mice immunized twice with 50,000 <i>Pyfabb/f⁻</i> salivary gland sporozoites (right panel) or mock-immunized with salivary gland debris (left panel), challenged by HTVI with 25 µg of phCMV-<i>Py</i>Tmp21-Luc 30 days after the last immunization. Each group contained 5 mice. (B) Inhibition of luciferase signal over the course of the experiment calculated as percentage reduction vs. the mock-immunization control. The data represents 5 individually analyzed mice in each group and correspond to mean ± SD. (C) Quantification of luciferase signal (shown as total flux p per second) in mice primed with 50,000 <i>Pyfabb/f⁻</i> salivary gland sporozoites and boosted with gWIZ-<i>Py</i>Tmp21 (right panel), or mock-immunized and injected with gWIZ (left panel), challenged as described in part A. Each group contained 4 to 5 mice. (D) Inhibition of luciferase signal over the course of the experiment calculated as percentage reduction vs. the mock-immunization control. The data represents 5 individually analyzed mice in each group and correspond to mean ± SD. (E) Quantification of luciferase signal (shown as total flux p per second) in mice primed with gWIZ-<i>Py</i>Tmp21 and boosted with 50,000 <i>Pyfabb/f⁻</i> salivary gland sporozoites (right panel), or injected with gWIZ and mock-immunized (left panel), challenged as described in part A. Each group contained 5 mice. (F) Inhibition of luciferase signal over the course of the experiment calculated as percentage reduction vs. the mock-immunization control. The data represents 5 individually analyzed mice in each group and correspond to mean ± SD.</p

Elimination of hepatocytes that express <i>Py</i>CSP-Luc is mediated by CD8⁺ cells.

<p>(A) Luciferase signal in live mice immunized twice with 50,000 <i>Pyfabb/f⁻</i> salivary gland sporozoites (right panel) or mock immunized (left panel) with salivary gland debris, and treated 14 days later for two consecutive days with antibodies anti-CD8 (top panel), anti-CD4 (middle panels) or an equivalent amount of control rat IgG2b, before HTVI challenge with 25 µg of phCMV-<i>Py</i>CSP-Luc and imaged 7 d post challenge as described. Each group contained 5 mice. The scale indicates radiance expressed as p/s/cm²/sr. (B) Quantitation of the data shown in part A. Radiance is shown as total flux (p/s). The data represents 5 individually analyzed mice in each group and correspond to mean ± SD; significant differences between the mean of the mock vs. <i>Pyfabb/f⁻</i>-immunized mice for each treatment calculated using the Mann-Whitney test are indicated (** = <i>p</i><0.01). (C) Kinetics of inhibition of luciferase expression upon depletion of CD8⁺ (red circles) or CD4⁺ (blue squares) T cells, or mock depletion (black triangles), calculated as percentage reduction compared to the mock immunization control for each condition. The data represents 5 individually analyzed mice in each group, and correspond to mean ± SD; significant differences between the CD8 or CD4 T cell depletion vs. the mock depletion are indicated (** = <i>p</i><0.01).</p

Glycan Masking of <i>Plasmodium vivax</i> Duffy Binding Protein for Probing Protein Binding Function and Vaccine Development

<div><p>Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of <i>Plasmodium vivax</i> Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to <i>P. vivax</i> vaccine development.</p></div

Summary of PvDBP polymorphism, inhibitory epitopes, and residues impacting DARC binding.

<p>The sequence of the solved Sal1strain PvDBP variant crystal structure <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Batchelor1" target="_blank">[13]</a> is shown. Polymorphic amino acids from 129 PvDBP sequences <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Gosi1" target="_blank">[28]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Xainli1" target="_blank">[29]</a> are listed below the Sal1 sequence. Alpha helices in the PvDBP crystal structure <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Batchelor1" target="_blank">[13]</a> are indicated by “h” and labeled helix 1a to helix 9 according to convention <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Hodder1" target="_blank">[71]</a>. Circles above the line-up indicate important residues. N-glycosylation sites are numbered according to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat-1003420-t001" target="_blank">Table 1</a> and colored green (wild type), blue (STBP glycan), orange (P1 and Max), and red (Max). Dimer interface <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Batchelor1" target="_blank">[13]</a> – black circles; polymorphism – light grey (rare, <10% of sequences), dark grey (>10% of sequences); mutations that effect DARC binding from this study and others <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Batchelor1" target="_blank">[13]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Hans1" target="_blank">[17]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Bolton1" target="_blank">[19]</a>, are colored blue (no effect), yellow with black shadowing (minor), orange (moderate), red (major), and black, differences between studies (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420.s007" target="_blank">Table S1</a>); linear epitopes targeted by inhibitory antibodies <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat.1003420-Chootong1" target="_blank">[20]</a> – black or grey shading (low inhibitory), blue shading (medium inhibitory), red shading (high inhibitory).</p

Effect of DARC phenotype on antibody binding inhibitory activity.

<p>COS-7 cells expressing PvDBPII as a GFP fusion protein were incubated with immune plasma and then RBC expressing the FyA (A) or FyB (B) Duffy blood group antigen were added. The inset shows that FyA RBCs (A) gave smaller rosettes than FyB RBCs (B) in the COS-7 cell–RBC binding assay. Statistical testing and p values as explained in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003420#ppat-1003420-g006" target="_blank">Figure 6B</a>.</p