14 research outputs found
Expression of full-length antibodies from recombinant AAV vectors.
<p>Levels of expressed IgG or immunoadhesin were analyzed by Western Blot after transfection of HEK293T cells with equal amounts of plasmid DNA (0.5 μg + 0.5 μg or 1 μg). Comparison of secreted (<b>A</b>) 4L6 IgGs or (<b>B</b>) 5L7 IgGs from co-transfection of heavy and light chain vectors (two vector approach) <i>vs</i>. transfection of bicistronic vectors (one vector approach). The two vector approach yielded slightly higher levels of secreted antibodies than the one vector approach. IgG1 versions of the 4L6 and 5L7 full-length antibodies expressed better than IgG2 versions.</p
Coomassie staining of purified antibodies and immunoadhesins.
<p>Purity and integrity of purified IgGs and immunoadhesins was verified by coomassie staining following large-scale transfection of HEK293T cells with ssAAV-IgG and scAAV-immunoadhesin vector plasmids. SDS-PAGE (1 μg of purified protein per lane) and staining under (<b>A</b>) non-reducing and (<b>B</b>) reducing conditions. Both conditions confirmed the expected size and composition of the tested proteins. The immunoadhesins and full-length IgG versions of 4L6 and 5L7 have unusually long heavy chain CDR3 regions compared to polyclonal rhesus IgG heavy chains, thus, heavy chains of 4L6 and 5L7 have a considerably higher MW.</p
Further improvements of ssAAV vector expression cassettes.
<p>The exact same samples were analyzed by two methods. Yields of secreted 5L7 IgG antibodies were compared by (<b>A</b>) Western Blot and quantified by (<b>B</b>) ELISA, following transfection of HEK293T cells with different ssAAV vector plasmids.</p
Levels of IgG expression from modified ssAAV vectors.
<p>Levels of expressed 5L7 IgG2 were analyzed by Western Blot after transfection of HEK 293T cells with equal amounts of bicistronic ssAAV vectors (1 μg). (<b>A</b>) The conventional ssAAV-5L7 IgG2 vector (as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158009#pone.0158009.g001" target="_blank">Fig 1B</a>) was modified by addition or deletion of peptides (SGSG, V5, Furin) and compared to each other. While all modifications improve expression of the antibody, only the SGSG version of the vector mediates correct F2A-Furin cleavage comparable to the conventional bicistronic ssAAV vector. (<b>B</b>) Demonstration of Furin-mediated cleavage of the F2A peptide remaining on the IgG heavy chain. Deletion of Furin peptide prevents removal of redundant amino acids from the heavy chain C-terminus following F2A cleavage.</p
Expression of 5L7 IgG1 after AAV-mediated transduction <i>in vitro</i>.
<p>AAV vectors were encapsidated with AAV1 wild-type (wt) capsid or AAV1 mutant capsids (Y445F and/or Y731F); in the case of ssAAV, we utilized the modified ssAAV vector construct containing both SGSG and WPRE. Purified AAV virus particles were then used for transduction. HEK293T cells were infected with (<b>A</b>) 2x10<sup>4</sup> rAAV genome copies per cell (GC/cell), (<b>B</b>) 5x10<sup>3</sup> GC/cell and (<b>C</b>) 1x10<sup>3</sup> GC/cell. (<b>D</b>) Rhesus fibroblast cells were infected with 2x10<sup>5</sup> GC/cell. AAV transduction experiments shown in (<b>A</b> + <b>D</b>) were conducted at a different time than experiments in (<b>B</b> + <b>C</b>). Levels of secreted antibody were measured by ELISA following the time of transduction. Values are depicted as mean ± SD (n = 3/group); ****p < 0.0001, ***<i>p</i> < 0.001, **<i>p</i> < 0.01, *<i>p</i> < 0.05.</p
Schematic illustration of AAV constructs and transgene products.
<p>Design of recombinant AAV vectors expressing antibody or antibody-like molecules. (<b>A</b>) Self-complementary AAV (scAAV) containing an expression cassette for a single-chain fragment variable immunoadhesin (scFvi). Upon expression, the scFvi dimerizes to form a mature immunoadhesin with a MW of approximately 120 kDa. The expression cassette is flanked by AAV2 inverted terminal repeats (ITRs); the 5' ITR is truncated to form double-stranded AAV genomes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158009#pone.0158009.ref043" target="_blank">43</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158009#pone.0158009.ref052" target="_blank">52</a>]. (<b>B</b>) Two strategies for achieving expression of full-length antibodies. The first approach, called the two vector approach, requires two scAAV vectors, one encoding IgG heavy chain and one the light chain. The second strategy, called the one vector approach, utilizes one single-stranded AAV (ssAAV) vector only, with heavy and light chains of IgG expressed from one open reading frame. The two polypeptide chains are separated by a 2A peptide from foot-and-mouth-disease virus (F2A) that mediates cleavage and a furin peptide that allows removal of redundant amino acids at the heavy chain C-terminus following furin enzyme-dependent cleavage. Thus, the heavy chain C-terminus is believed to attain an authentic sequence. The light chain N-terminus is believed to gain an authentic sequence following signal peptide (SP)-mediated cleavage. The transgene cassette is flanked by AAV2 ITRs to form single-stranded AAV genomes. The full-length authentic IgG has a MW of ≥ 150 kDa. Abbreviations: 5'ITRΔtrs, 5' inverted terminal repeat devoid of the terminal resolution site; Short CMV, a shortened variant of the immediate early CMV promoter (CMV) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158009#pone.0158009.ref016" target="_blank">16</a>]; SV40 intron, an intron from simian virus 40; SP, signal peptide; VL, variable light domain; L, serine-glycine linker peptide; VH, variable heavy domain; H, hinge region; CH, constant heavy domain; CL, constant light domain; pA, polyadenylation signal; Furin, cleavage sequence for the cellular protease furin.</p
Repeated low-dose SIVmac239 challenge of AAV-immunized and control animals.
<p>(<b>A</b>) Kaplan-Meier analysis of the three test groups. The percentage of animals remaining uninfected is plotted against the number of SIV exposures. SIV virus challenges were conducted in 3-week intervals. Four low-dose challenges with one half-maximal infectious dose (1x ID50) were followed by a two ID50 (2x) and a subsequent ten ID50 (10x) challenge. The survival curves are not significantly different (Mantel-Cox test). (<b>B</b>) Peak viral load comparison. The geometric mean (red line) is significantly different in the 5L7 IgG1 group compared to the control group (two-tailed, unpaired <i>t</i> test). (<b>C</b>) Set-point viral load analysis. All values within one group between weeks 8 and 25 were averaged, the resulting values were logarithmized (log10) and compared to each other. The geometric mean of the 5L7 IgG1 group differs significantly from the other two groups (two-tailed, unpaired <i>t</i> test). (<b>D</b>) Time of peak viremia. Most of the immunized animals show peak viremia at week 3, while 5 of 6 controls peak at week 2. (<b>E</b> and <b>F</b>) Viral loads <i>in vivo</i> after infectious exposure with SIVmac239. Viral loads in plasma of AAV animals (blue) and controls (red) measured as SIV RNA genome equivalent per ml are shown as a function of time since the infectious exposure. Control animal 139–08 was a rapid progressor and had to be sacrificed after 9 weeks due to AIDS-related symptoms. Test animal 84–05 remained uninfected after 6 SIV challenges.</p
Neutralization of SIV and binding of purified proteins <i>in vitro</i>.
<p>The dashed line indicates 50% RLU (relative light units) representing 50% neutralization activity against the tested SIV strain. Lowest RLU indicates highest neutralization. Binding of purified proteins was tested by a SIVmac239 gp140 ELISA, high absorbance indicates high binding. SPF serum (specific pathogen free) from naïve animals was used as negative control, a pool of antisera from SIV-infected animals served as positive control. (<b>A</b>) None of the antibodies or immunoadhesins showed neutralization against the resistant virus strain SIVmac239 tested at 50 μg/ml. Similar results were obtained in both the SEAP and TZMbl assays. (<b>B</b>) Neutralization curve of SIVmac239 with 5L7 IgG1 starting at 1mg/ml. (<b>C</b>) All of the tested antibodies and immunoadhesins showed equivalent neutralization activity against SIVmac316 with an average IC50 (half-maximal inhibitory concentration) of 0.002 μg/ml. (<b>D</b>) Animal sera from week 10 after rAAV administration (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005090#ppat.1005090.g001" target="_blank">Fig 1A</a>) were diluted to 4 μg/ml based on previous ELISA quantitations, and tested for neutralizing activity against SIVmac316. The average IC50 measured corresponds to the IC50 of the purified proteins. (<b>E</b>) Animal sera of 84–05 from weeks 1 and 12 after AAV administration lacked detectable neutralizing activity against SIVmac239. (<b>F</b>) Antibodies and immunoadhesins were tested for their ability to bind SIVmac239 gp140. All of the antibody constructs showed equivalent binding activity as determined by absorbance at 450 nm.</p
Serum concentration of the IgG1 mAbs 4L6 and 5L7, and emerging anti-mAb responses following recombinant AAV administration.
<p>Levels of produced antibodies were measured in sera from immunized animals over time by a gp140 capture ELISA. SIV challenge is indicated by arrows. (<b>A</b>) Animals that received rAAV-5L7 IgG1. 84–05, 153–10 and 157–10 were given one ssAAV vector expressing both heavy and light chain of the antibody 5L7 IgG1. 111–09, 120–09 and 154–10 were given an equal mixture of two scAAV vectors expressing either heavy chain or light chain of the antibody 5L7 IgG1. (<b>B</b>) Animals that received rAAV-4L6 IgG1. All six animals were given one ssAAV vector expressing both heavy and light chain of the antibody 4L6 IgG1. (<b>C</b> and <b>D</b>) Antibody responses to 5L7 IgG1 and 4L6 IgG1, respectively, following recombinant AAV administration. Humoral immune responses were measured in sera from immunized animals over time by ELISA. The reactivity of serum was tested against homologous purified protein using a conjugated anti-lambda secondary antibody for detection since both 5L7 IgG1 and 4L6 IgG1 bear a kappa light chain. (<b>E</b>) Reactivity of sera from 5L7 IgG1 recipients to purified mAbs and immunoadhesins at week 10 after AAV administration. (<b>F</b>) Reactivity of sera from 4L6 IgG1 animals to purified mAbs and immunoadhesins at week 6 after AAV administration.</p
A human inferred germline antibody binds to an immunodominant epitope and neutralizes Zika virus
<div><p>The isolation of neutralizing monoclonal antibodies (nmAbs) against the Zika virus (ZIKV) might lead to novel preventative strategies for infections in at-risk individuals, primarily pregnant women. Here we describe the characterization of human mAbs from the plasmablasts of an acutely infected patient. One of the 18 mAbs had the unusual feature of binding to and neutralizing ZIKV despite not appearing to have been diversified by affinity maturation. This mAb neutralized ZIKV (Neut<sub>50</sub> ~ 2 μg/ml) but did not react with any of the four dengue virus serotypes. Except for the expected junctional diversity created by the joining of the V-(D)-J genes, there was no deviation from immunoglobulin germline genes. This is a rare example of a human mAb with neutralizing activity in the absence of detectable somatic hypermutation. Importantly, binding of this mAb to ZIKV was specifically inhibited by human plasma from ZIKV-exposed individuals, suggesting that it may be of value in a diagnostic setting.</p></div