Refinement of immunizing antigens to produce functional blocking antibodies against the AniA nitrite reductase of <i>Neisseria gonorrhoeae</i>

<div><p>The emergence of multi-drug resistant <i>Neisseria gonorrhoeae</i> has generated an urgent need for novel therapies or a vaccine to prevent gonococcal disease. In this study we investigate the potential of targeting the surface exposed nitrite reductase, AniA, to block activity by producing functional blocking antibodies. AniA activity is essential for anaerobic growth and biofilm formation of <i>N</i>. <i>gonorrhoeae</i> and functional blocking antibodies may prevent colonisation and disease. Seven peptides covering regions adjacent to the active site were designed based on the AniA structure. Six of the seven peptide conjugates generated immune responses. Peptide 7, GALGQLKVEGAEN, was able to elicit antibodies capable of blocking AniA activity. Antiserum raised against the peptide 7 conjugate detected AniA in 20 <i>N</i>. <i>gonorrhoeae</i> clinical isolates. Recombinant AniA protein antigens were also assessed in this study and generated high-titre, functional blocking antibody responses. Peptide 7 conjugates or truncated recombinant AniA antigens have potential for inclusion in a vaccine against <i>N</i>. <i>gonorrhoeae</i>.</p></div

Analysis of the murine immune response against the synthetic peptides.

<p>The titres of pooled pre-immune and pooled post-immune sera from each group of mice were determined by ELISA against purified recombinant AniA (antigen 4 –see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182555#pone.0182555.g002" target="_blank">Fig 2</a> and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182555#pone.0182555.ref008" target="_blank">8</a>]) and are shown as the geometric mean titre of triplicate assays. Western blot analyses were performed with pooled post-immune sera from each group of mice against whole cell lysates of <i>N</i>. <i>gonorrhoeae</i> 1291 wild-type and <i>aniA</i>::kan. Pooled pre-immune sera from each group of mice were also analysed by western blotting and no reactivity against AniA was observed (data not shown).</p

Plasmids, strains and primers used in this study.

<p>Plasmids, strains and primers used in this study.</p

Analysis of the immunogenicity of the synthetic peptides and recombinant antigens 1 and 5 in rabbits.

<p>The titres of the pre-immune and post-immune sera from each rabbit were determined by ELISA against purified recombinant AniA (antigen 4 –see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182555#pone.0182555.g002" target="_blank">Fig 2</a> and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182555#pone.0182555.ref008" target="_blank">8</a>]) and are shown as the geometric mean titre of triplicate assays. Western blot analyses were performed with post-immune sera from each rabbit against whole cell lysates of <i>N</i>. <i>gonorrhoeae</i> 1291 wild-type and <i>aniA</i>::kan. Pre-immune serum from each rabbit was also analysed by western blotting and no reactivity against AniA was observed (data not shown).</p

Analysis of the murine immune response of the recombinant antigens 1–8.

<p>The titres of post-immune sera from individual mice in each group were determined by ELISA against <i>N</i>. <i>gonorrhoeae</i> 1291 wild-type and <i>aniA</i>:<i>kan</i> whole cells performed in triplicate. Titres are shown as the geometric mean titres of the means from the triplicate individual assays.</p

Diagrammatic representation of the full-length AniA protein from <i>N</i>. <i>gonorrhoeae</i> 1291 and of the recombinant AniA proteins (antigens 1–8) with various truncations of the N- and C-termini used for immunization.

<p>Lipid modification site = signal peptide containing a typical lipoprotein processing site (ALAAC), similar to sequences found in two other outer membrane gonococcal lipoproteins Lip/H.8 and Laz. This sequence is cleaved between Ala and Cys residues by signal peptidase II followed by N-terminal acylation of the Cys residue with palmitic acid (1–10, red); N-terminal repeat region = high in Ala and Pro content and contains several imperfect repeats of the AAEAP motif found in Lip and Laz [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182555#pone.0182555.ref009" target="_blank">9</a>] (11–44, green); Nitrite reductase = region included in the crystal structure of <i>N</i>. <i>gonorrhoeae</i> AniA and contains residues essential for copper binding at the type I site and type II site as well as the highly conserved active site residues Asp121 and His262 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182555#pone.0182555.ref017" target="_blank">17</a>] (45–338, black); C-terminal glycosylation region, = contains the glycosylated serine residues, also contains four direct contiguous copies of a pentapeptide repeat, AASAP (339–384, blue).</p

Analysis of the surface expression of recombinant AniA in AniA-BL21 by whole cell ELISA.

<p>Whole cell ELISA was performed on pET-BL21 and AniA-BL21 cells induced for protein expression at 22°C for 3 hours using antiserum raised against recombinant AniA antigen 4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182555#pone.0182555.ref008" target="_blank">8</a>] two-fold serially diluted starting at a dilution of 1: 1000. Polyclonal Goat Anti-Rabbit Immunoglobulin HRP-conjugated (DakoCytomation) was used as the secondary antibody at a dilution of 1: 10 000. Bars represent the mean from triplicate assays. Error bars represent ±1SD from the mean.</p

AniA activity blocking assays.

<p>(A) Bars represent AniA nitrite reductase activity expressed as the mean reaction rate (nmoles nitrite reduced min^-1 ml^-1 OD of cells^-1) from triplicate assays of AniA-BL21 cells pre-incubated with a 1:20 dilution of pooled pre-immune sera or post-immune sera from each rabbit immunized with peptides 1–7, antigen 1 or antigen 5. Statistical significance was determined using a two-tailed unpaired Student’s <i>t-test</i>. * = <i>P</i><0.05; ** = <i>P</i><0.005. (B) Bars represent nitrite reductase activity from triplicate assays of <i>N</i>. <i>gonorrhoeae</i> whole cells pre-incubated with a 1:20 dilution of pooled pre-immune sera or post-immune serum from the rabbit immunized with peptide 7. Error bars represent ±1SD from the mean. Statistical significance was determined using a one-tailed unpaired Student’s <i>t-test</i>. * = <i>P</i><0.05.</p

Crystal structure of the AniA trimer [17].

<p>(A) The surface structure of the AniA trimer. The seven peptides that were designed to generate antibodies to inhibit AniA function are shown in seven different colours; 1 (maroon), 2 (cyan), 3 (green), 4 (yellow), 5 (purple), 6 (magenta), 7 (red). (B) Each monomer is shown in three different shades of grey. (C) The active sites of the AniA protein. Orange sphere indicates coppers (Cu). The inset shows the type I and type II Cu sites.</p

Antiserum raised against peptide 7 is cross-reactive with AniA from a range of <i>N</i>. <i>gonorrhoeae</i> clinical isolates.

<p>Antiserum from the rabbit immunized with peptide 7 was analysed by western blotting against cell lysates from a range of <i>N</i>. <i>gonorrhoeae</i> clinical isolates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182555#pone.0182555.t001" target="_blank">Table 1</a>) at a dilution of 1: 5000. M = molecular weight marker, 1: 1291, 2: 1291 <i>aniA</i>::kan, 3: 02G0142, 4: 90/G747, 5: 98G1131, 6: 88G285, 7: 02D004, 8: 02D156, 9: 02W001, 10: 02W006, 11: 98D159, 12: 97D040, 13: 01D1052, 14: 01G1370, 15: 02G0427, 16: 02G1036, 17: 94G163, 18: 00G0794, 19: 01D064, 20: 01D100, 21: 97D059, 22: 96D551.</p