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. 2015 Jun;93(6):619-31.
doi: 10.1007/s00109-015-1280-4. Epub 2015 May 5.

Retargeting pre-existing human antibodies to a bacterial pathogen with an alpha-Gal conjugated aptamer

Sascha A Kristian  1 , John H Hwang, Bradley Hall, Emma Leire, John Iacomini, Robert Old, Uri Galili, Charles Roberts, Kary B Mullis, Mike Westby, Victor Nizet
Affiliations

Retargeting pre-existing human antibodies to a bacterial pathogen with an alpha-Gal conjugated aptamer

Sascha A Kristian et al. J Mol Med (Berl). 2015 Jun.

Abstract

The ever-increasing threat of multi-drug resistant bacterial infections has spurred renewed interest in alternative approaches to classical antibiotic therapy. In contrast to other mammals, humans do not express the galactose-α-1,3-galactosyl-β-1,4-N-acetyl-glucosamine (α-Gal) epitope. As a result of exposure of humans to α-Gal in the environment, a large proportion of circulating antibodies are specific for the trisaccharide. In this study, we examine whether these anti-Gal antibodies can be recruited and redirected to exert anti-bacterial activity. We show that a specific DNA aptamer conjugated to an α-Gal epitope at its 5' end, herein termed an alphamer, can bind to group A Streptococcus (GAS) bacteria by recognition of a conserved region of the surface-anchored M protein. The anti-GAS alphamer was shown to recruit anti-Gal antibodies to the streptococcal surface in an α-Gal-specific manner, elicit uptake and killing of the bacteria by human phagocytes, and slow growth of invasive GAS in human whole blood. These studies provide a first in vitro proof of concept that alphamers have the potential to redirect pre-existing antibodies to bacteria in a specific manner and trigger an immediate antibacterial immune response. Further validation of this novel therapeutic approach of applying α-Gal technology in in vivo models of bacterial infection is warranted.

Key messages: . α-Gal-tagged aptamers lead to GAS opsonization with anti-Gal antibodies. . α-Gal-tagged aptamers confer phagocytosis and killing of GAS cells by human phagocytes. . α-Gal-tagged aptamers reduces replication of GAS in human blood. . α-Gal-tagged aptamers may have the potential to be used as novel passive immunization drugs.

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Conflict of interest statement

Conflict of interest The aptamer work was funded in full by Altermune Technologies, LLC (Irvine, CA). Several of the authors are employees, have executive roles and/or are consultants to Altermune Technologies, LLC with respect to their efforts to develop aptamers as therapeutic drugs.

Figures

Fig. 1
Fig. 1
Initial binding tests with DNA aptamer 20A24P and live GAS cells. a Stationary phase GAS SF370 or GAS 5448 cells were incubated for 45 min at 37 °C with the indicated end concentrations of 5′- or 3′-FAM labeled 80-nucleotide (nt) GAS aptamer 20A24P (solid black lines), the 80-nt negative control aptamer 5′-FAM-RAND-80 (dashed lines), or aptamer vehicle (gray solid). Subsequently, the bacteria were washed and subjected to flow cytometry to measure the fluorescence intensities of 20,000 bacterial particles per sample. b Performed as above, GAS 5448 incubated with 5′-FAM labeled GAS aptamer 20A24P over a range of concentrations. The histograms show FL-1 channel overlays for representative samples of several experiments performed. Each sample was run in duplicate with similar results
Fig. 2
Fig. 2
Truncation of GAS aptamer 20A24P from 80 to 52 nucleotides leads to enhanced binding to live GAS 5448 cells. a Binding of the indicated 5′-FAM labeled truncated versions of the GAS aptamer 20A24P (solid black lines) and 39-nt negative control aptamer RAND-39 (dashed lines) at 500 nM end concentration or aptamer vehicle (gray solid). For the data shown for 20A24P.A9, 3′-FAM labeled GAS aptamer (solid line) and 52-nt negative control aptamer RAND-52 (dashed line) were used. The length of the respective GAS aptamers is indicated in parentheses. b Binding comparison of parental 5′-FAM 80-nt GAS aptamer 20A24P (black solid lines) and 5′-FAM truncated 52-nt aptamer 20A24P.A3 (dashed lines) at the indicated aptamer end concentrations vs. aptamer vehicle (gray solid). For all samples, the bacteria were incubated with the aptamers or vehicle for 37 °C at 45 min, then washed and subjected to flow cytometry to measure the fluorescence intensities of 20,000 bacterial particles per sample. Each sample was run in duplicate with similar results
Fig. 3
Fig. 3
Growth phase dependence of 20A24P.A3 binding to live GAS cells. GAS 5448 cells were grown in THB media to the indicated optical densities at 600 nm (OD600), then washed and photometrically adjusted to the same bacterial concentrations. Then, the binding of 5′-FAM versions of the 52-nt GAS aptamer 20A24P.A3 (black solid lines) to the bacteria was tested as described in Figs. 1 and 2. 5′-FAM 52-nt aptamer RAND-52 (dashed lines) and aptamer vehicle (gray solid) served as negative controls. FL-1 histogram overlays for the indicated stages of growth for a representative experiment of several performed are shown. For each experiment, the samples were run in duplicate yielding similar results
Fig. 4
Fig. 4
M protein is the target of aptamer 20A24P on GAS cells. a The binding of 5′-FAM GAS aptamer 20A24P (black solid lines) at a concentration of 500 nM to stationary phase cells of live GAS 5448 wild type, an isogenic M1-deficient emm1 mutant, and a complemented emm1 mutant was determined by flow cytometry. Five hundred nanomolar 5′-FAM 80-nt aptamer RAND-80 (dashed lines) and aptamer vehicle (gray solid) served as negative controls. The histogram overlays show the FL-1 fluorescence intensities of 20,000 bacterial particles per sample. b By ELISA, the binding of 3′-biotinylated 20A24P to immobilized recombinant full-length mature M1 protein and truncated versions, which are schematically depicted, was tested. Biotinylated aptamer RAND-80 and vehicle served as negative controls. The photographs show representative results of several experiments performed. Yellow color indicates binding of an aptamer to the respective protein, whereas colorless wells indicate lack of binding of a given test reagent to the immobilized proteins
Fig. 5
Fig. 5
Recognition of 5′-α-Gal GAS alphamers on bacteria by anti-α-Gal antibodies. 5′-α-Gal conjugated versions of the GAS aptamers 20A24P (α20A24P) and 20A24P.A3 (α20A24P.A3) as well as negative control RAND-80 (αRAND-80), herein referred to as alphamers, were prepared. a Binding of 5′-α-Gal, 3′-FAM α20A24P (black solid lines) and αRAND-80 (dashed lines) to live GAS 5448 and SF370 cells at an end concentration of 1 μM. The binding of transgenic mouse IgM (b, d) and IgG (c) antibodies to alphamer coated, live GAS 5448 cells was tested by flow cytometry. Bacteria were pre-incubated with 5′-α-Gal, 3′-FAM-labeled GAS alphamer α20A24P (Black solid lines in b, c), non-FAM-labeled 20A24P.A3 (black solid lines in d), 3′-FAM-labeled control alphamer αRAND-80 (dashed lines) or alphamer vehicle (gray solid). The binding of the primary antibodies was detected with secondary Alexa Fluor® 647 anti-mouse IgM (b, d) or anti-mouse IgG (c) antibodies. This allowed for simultaneous visualization of alphamer and mouse antibody binding in the FL-1 and FL-4 channels, respectively. Samples incubated with alphamers but no mouse IgM or IgG (–mouse IgM; –mouse IgG) served as negative controls to rule out unspecific secondary antibody binding to the bacteria. Representative results of several experiments performed are shown in the histogram overlays. e To determine if the α-Gal portion was critical for mouse antibody binding to 5′-FAM-α20A24P-coated GAS 5448, the ability of the alphamer and non-α-Gal-conjugated aptamer 5′-FAM-20A24P at 1 μM endconcentration to redirect mouse IgM antibodies to the bacteria was compared as above. Vehicle and 5′-FAM-αRAND-80 were used as controls. FL-1 (= Aptamer/alphamer binding) vs. FL-4 (= IgM binding) dot blots for one representative experiment of several performed are shown
Fig. 6
Fig. 6
GAS alphamers confer uptake of GAS M1T1 cells by human phagocytes in the presence of mouse and human polyclonal IgG in an alpha-Gal-specific manner. Green fluorescent GAS cells were pre-incubated with the indicated 5′-α-Gal conjugated GAS alphamers, non-alpha-Gal GAS aptamer, the respective control alphamer/aptamer, or vehicle only. Then, polyclonal mouse IgG or human IgG (hIVIG) and, finally, purified human blood neutrophils were added to the bacteria. After 20 min of co-incubation, the phagocytes were washed and examined for uptake of GAS by fluorescence microscopy. Extracellular bacteria were distinguishable from intracellular bacteria by counterstaining with ethidium bromide. Samples were run in quintuplicate or hexuplicate, and average percentages of neutrophils with phagocytosed bacteria ± SD are shown in the graphs. ***p<0.00005, unpaired t test, GAS alphamers vs. respective control alphamers and vehicle controls. Representative data of one experiment for each data set of at least two performed are shown
Fig. 7
Fig. 7
Activity of GAS alphamer in opsonophagocytic killing assays and whole blood killing assay. a Late exponential phase GAS SF370 was pre-incubated with GAS alphamer α20A24P or control alphamer αRAND-80. After 20 min, neutrophils and human IgG (hIVIG) were added. Samples to which vehicle (HBSS+/+) and hIVIG instead of neutrophils were added served as no phagocyte controls. At various time points, the percentage of live colony forming units as compared to time point 0 min was determined. Quadruplicate to hexuplicate samples were run for each condition, and average bacterial percentage values as compared to the initial bacterial concentrations±SD plotted. *p<0.05; ***p<0.001, α20A24P vs. αRAND-80 in presence of neutrophils; two-way ANOVA with Bonferroni post test. b 1.1×105 CFU/mL of GAS M1T1 5448 were pre-incubated for 15 min with 1 μM of the GAS alphamer α20A24P (closed bars) or the control alphamer αRAND-80 (open bars) in a total volume of 30 μL. Then, 120 μL of human blood was added. Thus, the alphamer end concentrations were 200 nM. After 60, 120, 180, and 240 min of co-incubation, the bacterial concentrations were determined. Each sample was run in quadruplicate and average values± SD are plotted. *p<0.05, α20A24P vs. αRAND-80 two-way ANOVA with Bonferroni post tests
Fig. 8
Fig. 8
Summary scheme for characterizing aptamer-based drugs. In a process termed Systematic Evolution of Ligands by Exponential Enrichment (SELEX), bacterial whole cells or bacterial components, aptamer libraries, PCR, and sequencing are used to identify aptamer candidates for further characterization. Fluorescently tagged aptamer candidates are synthesized and prioritized based on their binding to several strains of the target bacterial species by flow cytometry. Then, alphamer versions of prioritized aptamers are prepared. After confirmation that the alphamers bind to bacteria and attract anti-Gal antibodies to the microbial surface, alphamer activity is tested in correlate of protection assays in vitro (complement deposition and lysis, phagocytosis assays) that may predict the in vivo activity of candidate molecules. Alphamers with strong in vitro activity could then be tested in mouse infection models and later in humans
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