BRED: A Simple and Powerful Tool for Constructing Mutant and Recombinant Bacteriophage Genomes

Advances in DNA sequencing technology have facilitated the determination of hundreds of complete genome sequences both for bacteria and their bacteriophages. Some of these bacteria have well-developed and facile genetic systems for constructing mutants to determine gene function, and recombineering is a particularly effective tool. However, generally applicable methods for constructing defined mutants of bacteriophages are poorly developed, in part because of the inability to use selectable markers such as drug resistance genes during viral lytic growth. Here we describe a method for simple and effective directed mutagenesis of bacteriophage genomes using Bacteriophage Recombineering of Electroporated DNA (BRED), in which a highly efficient recombineering system is utilized directly on electroporated phage DNA; no selection is required and mutants can be readily detected by PCR. We describe the use of BRED to construct unmarked gene deletions, in-frame internal deletions, base substitutions, precise gene replacements, and the addition of gene tags

Safety and immunogenicity of heterologous versus homologous prime-boost schedules with an adenoviral vectored and mRNA COVID-19 vaccine (Com-COV): a single-blind, randomised, non-inferiority trial

Background: Use of heterologous prime-boost COVID-19 vaccine schedules could facilitate mass COVID-19 immunisation. However, we have previously reported that heterologous schedules incorporating an adenoviral vectored vaccine (ChAdOx1 nCoV-19, AstraZeneca; hereafter referred to as ChAd) and an mRNA vaccine (BNT162b2, Pfizer–BioNTech; hereafter referred to as BNT) at a 4-week interval are more reactogenic than homologous schedules. Here, we report the safety and immunogenicity of heterologous schedules with the ChAd and BNT vaccines. Methods: Com-COV is a participant-blinded, randomised, non-inferiority trial evaluating vaccine safety, reactogenicity, and immunogenicity. Adults aged 50 years and older with no or well controlled comorbidities and no previous SARS-CoV-2 infection by laboratory confirmation were eligible and were recruited at eight sites across the UK. The majority of eligible participants were enrolled into the general cohort (28-day or 84-day prime-boost intervals), who were randomly assigned (1:1:1:1:1:1:1:1) to receive ChAd/ChAd, ChAd/BNT, BNT/BNT, or BNT/ChAd, administered at either 28-day or 84-day prime-boost intervals. A small subset of eligible participants (n=100) were enrolled into an immunology cohort, who had additional blood tests to evaluate immune responses; these participants were randomly assigned (1:1:1:1) to the four schedules (28-day interval only). Participants were masked to the vaccine received but not to the prime-boost interval. The primary endpoint was the geometric mean ratio (GMR) of serum SARS-CoV-2 anti-spike IgG concentration (measured by ELISA) at 28 days after boost, when comparing ChAd/BNT with ChAd/ChAd, and BNT/ChAd with BNT/BNT. The heterologous schedules were considered non-inferior to the approved homologous schedules if the lower limit of the one-sided 97·5% CI of the GMR of these comparisons was greater than 0·63. The primary analysis was done in the per-protocol population, who were seronegative at baseline. Safety analyses were done among participants receiving at least one dose of a study vaccine. The trial is registered with ISRCTN, 69254139. Findings: Between Feb 11 and Feb 26, 2021, 830 participants were enrolled and randomised, including 463 participants with a 28-day prime-boost interval, for whom results are reported here. The mean age of participants was 57·8 years (SD 4·7), with 212 (46%) female participants and 117 (25%) from ethnic minorities. At day 28 post boost, the geometric mean concentration of SARS-CoV-2 anti-spike IgG in ChAd/BNT recipients (12 906 ELU/mL) was non-inferior to that in ChAd/ChAd recipients (1392 ELU/mL), with a GMR of 9·2 (one-sided 97·5% CI 7·5 to ∞). In participants primed with BNT, we did not show non-inferiority of the heterologous schedule (BNT/ChAd, 7133 ELU/mL) against the homologous schedule (BNT/BNT, 14 080 ELU/mL), with a GMR of 0·51 (one-sided 97·5% CI 0·43 to ∞). Four serious adverse events occurred across all groups, none of which were considered to be related to immunisation. Interpretation: Despite the BNT/ChAd regimen not meeting non-inferiority criteria, the SARS-CoV-2 anti-spike IgG concentrations of both heterologous schedules were higher than that of a licensed vaccine schedule (ChAd/ChAd) with proven efficacy against COVID-19 disease and hospitalisation. Along with the higher immunogenicity of ChAd/BNT compared with ChAD/ChAd, these data support flexibility in the use of heterologous prime-boost vaccination using ChAd and BNT COVID-19 vaccines. Funding: UK Vaccine Task Force and National Institute for Health Research

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

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. 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. 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. 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. 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

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

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. 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. 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. 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

Both forms of gp33 bind similarly to the BPs <i>33–34</i> intergenic region.

<p>(A) EMSA analysis shows that gp33¹⁰³ binds to the <i>33</i>–<i>34</i> intergenic region with a complicated pattern of binding with four distinct complexes, labeled C1, C2, C3, and C4. No binding was seen between gp33¹⁰³ and a non-speciic DNA substrate. Protein concentrations are as follows: 1) none 2) 0.16 μM 3) 0.54 μM, 4) 1.6 μM, 5) 5.4 μM, 6) 16 μM, 7) 54 μM. (B) DNA binding assays show that both gp33¹⁰³ and gp33¹³⁶ bind similarly to a DNA substrate containing PCR amplified DNA from the BPs gene <i>33–34</i> intergenic region (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.s002" target="_blank">S1 Table</a>). Protein concentrations are as shown in A. (C) Size-exclusion chromatograms for gp33¹⁰³ (orange) and the molecular weight markers (green; 66kDa, 29kDa, 12.4kDa, 6.5kDa) are overlayed. (D) The inset graph shows a standard plot for the molecular mass for the protein standards against the ration of their elution volume (Ve) and void volume (Vo) (blue diamonds); the molecular mass of gp33¹⁰³ is predicted to be 40.4kDa (orange diamond).</p

BPs gp33¹⁰³ binding to substrate variants of the <i>33</i>–<i>34</i> intergenic region derived from repressor-insensitive BPs mutants.

<p>(A) Locations of the mutations in each substrate (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.t001" target="_blank">Table 1</a>). (B) Binding profiles of gp33¹⁰³ to each <i>33</i>–<i>34</i> substrate. The concentrations of protein are as follows: 1) none, 2) 0.16μM, 3) 0.54μM, 4) 1.6μM, 5) 5.4μM, 6) 16μM, 7) 54μM. Protein affinities are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.t001" target="_blank">Table 1</a>.</p

BPs gp33¹⁰³ binds to two adjacent 12 bp palindromes.

<p>(A and B) EMSAs showing gp33¹⁰³ binding to substrates containing two 12bp palindromic half sites (yellow boxes) spaced either 8 bp (panel A) or 5 bp apart (panel B). Substrates in which one half site is mutationally ablated is shown as a white box.</p

BPs gp33¹⁰³ bends DNA but does not facilitate loop formation.

<p>(A) The 12bp of O_R was cloned into plasmid pBend2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.ref020" target="_blank">20</a>] using unique restriction enzyme sites SalI and XbaI to create pVMV22. Equal-sized fragments of DNA were excised from pVMV22 with O_R located at different positions relative to the edge of the probe by cleaving with appropriate enzymes. BPs gp33¹⁰³ (0.54μM) was incubated with each radiolabeled probe and run on a native polyacrylamide gel. The only gp33¹⁰³-DNA complex (C1) does not show any differences in relative mobility with different substrates. (B) A second plasmid containing the entire BPs <i>33–34</i> intergenic region (pVMV21) was analyzed similarly, suggesting that a bend of about 40° is introduced into the major complex (equivalent to C-4, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.g002" target="_blank">Fig 2A</a>) by gp33¹⁰³ binding. (C) To determine if intermolecular DNA bridges are formed by gp33¹⁰³, two different sized fragments of DNA containing the <i>33–34</i> intergenic region (366 bp and 256 bp) were PCR amplified and radiolabeled (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.s002" target="_blank">S1 Table</a>). The leftmost six lanes contain only the 366 bp substrate, the middle six lanes contain only the 256 bp substrate, and the rightmost six lanes contain both substrates. Complexes formed with each substrate are indicated by long and short arrows respectively. The concentration of protein in each series of substrates is 1) none, 2) 0.54μM, 3) 1.6μM, 4) 5.4μM, 5) 16μM, 6) 54 μM. Protein affinities are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.t001" target="_blank">Table 1</a>.</p

Complete and partial operator sites found throughout the BPs genome.

<p>(A) Map of the BPs genome showing open reading frames (colored boxes) with the positions of each of the operator sites indicated. (B) Sequences of the intergenic regions containing operators. Promoter sequences (green text), start codons of open reading frames (blue text), and the stop codons of open reading frames (red text) are shown. Operator half sites corresponding to the 12 bp sequence 5’-CGACATATGTCG are highlighted in yellow. Operator half sites containing sequence departures within these 12 bp sites are highlighted in orange or pink (reflecting gp33¹⁰³ binding or no observed binding respectively; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.g007" target="_blank">Fig 7</a>). O_Rep-R and O_R-L are highlighted in gray. Confirmed transcription start sites are marked as +1 [[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.ref013" target="_blank">13</a>] L. Oldfield and GFH, manuscript in preparation]. (D) Promoter fusions to a mCherry reporter gene were constructed in an integration-proficient vector, transformed into both <i>M</i>. <i>smegmatis</i> (blue bars) and a BPs lysogen (red bars) and fluorescence levels measured.</p

BPs gp33¹⁰³ binds with variable affinities to a series of end-deletion substrates.

<p>Substrates were PCR amplified from the BPs <i>33–34</i> intergenic region using primers located at regular intervals from each end to sequentially shorten the <i>33–34</i> intergenic region from each end (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.s002" target="_blank">S1 Table</a>). The sequences included in each substrate are shown as horizontal black lines either above or below a schematic representation of the <i>33</i>–<i>34</i> intergenic region. Genes 33 and 34 are represented by blue and orange arrows respectively, and O_R and O_Rep are shown in yellow. Protein concentrations are same as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.g002" target="_blank">Fig 2A</a>. Protein affinities are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137187#pone.0137187.t001" target="_blank">Table 1</a>.</p