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. 2016 Apr 27:6:24913.
doi: 10.1038/srep24913.

Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic Uremic Syndrome (HUS)

Maria P Mejías  1 , Yanina Hiriart  2   3 , Constanza Lauché  2   3 , Romina J Fernández-Brando  1 , Romina Pardo  2   3 , Andrea Bruballa  1 , María V Ramos  1 , Fernando A Goldbaum  2   3 , Marina S Palermo  1 , Vanesa Zylberman  2   3
Affiliations

Development of camelid single chain antibodies against Shiga toxin type 2 (Stx2) with therapeutic potential against Hemolytic Uremic Syndrome (HUS)

Maria P Mejías et al. Sci Rep. .

Abstract

Shiga toxin (Stx)-producing Escherichia coli (STEC) infections are implicated in the development of the life-threatening Hemolytic Uremic Syndrome (HUS). Despite the magnitude of the social and economic problems caused by STEC infections, no licensed vaccine or effective therapy is presently available for human use. Single chain antibodies (VHH) produced by camelids exhibit several advantages in comparison with conventional antibodies, making them promising tools for diagnosis and therapy. In the present work, the properties of a recently developed immunogen, which induces high affinity and protective antibodies against Stx type 2 (Stx2), were exploited to develop VHHs with therapeutic potential against HUS. We identified a family of VHHs against the B subunit of Stx2 (Stx2B) that neutralize Stx2 in vitro at subnanomolar concentrations. One VHH was selected and was engineered into a trivalent molecule (two copies of anti-Stx2B VHH and one anti-seroalbumin VHH). The resulting molecule presented extended in vivo half-life and high therapeutic activity, as demonstrated in three different mouse models of Stx2-toxicity: a single i.v. lethal dose of Stx2, several i.v. incremental doses of Stx2 and intragastrical STEC infection. This simple antitoxin agent should offer new therapeutic options for treating STEC infections to prevent or ameliorate HUS outcome.

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Figures

Figure 1
Figure 1. Stx2-neutralizing activity of VHH clones from different families.
Serially diluted purified VHHs were tested on the Vero cell neutralization assay as detailed in Materials and Methods. The percentage of cell survival, which is a measurement of the toxin neutralization, was calculated by the following formula: [(ODtoxin+VHH − ODtoxin only)/(ODno toxin − ODtoxin only)] × 100. Each sample was tested by triplicate and is represented as mean ± SEM. (A) VHH clones from family 1. Six representative clones from family 1 are shown. (B) VHH clones from other families. One representative clone from each family is shown.
Figure 2
Figure 2. In vitro activity of 2vb27 VHH under different formats.
(A) Serially diluted purified 2vb27, (2vb27)2 or (2vb27)2-SA were tested on the Vero cell neutralization assay as detailed in Materials and Methods. Each sample was tested by quadruplicate and is represented as mean ± SEM. (B) Binding of (2vb27)2-SA to human seroalbumin (SA). Nickel coated Maxisorp microtiter plates were incubated with serially diluted (2vb27)2-SA. Biotinylated human SA was added and binding was detected with Streptavidin-HRP. Reaction was developed with TMB and absorbance was read at 450 nm.
Figure 3
Figure 3. In vivo activity of 2vb27 VHH under different formats.
(A) Persistence in circulation of VHHs. Naïve mice (4 mice/group) were inoculated with 0.5 nmoles of 2vb27, (2vb27)2 or (2vb27)2-SA, and they were subsequently bled at the indicated time points. In vitro Stx2-neutralizing capacity of plasma samples (dilution 1/50) was measured on Vero cells as described in Materials and Methods. Each sample was tested by triplicate and is represented as mean ± SEM. ***p < 0.001 vs 2vb27, **p < 0.01 and *p < 0.05 vs 2vb27 and (2vb27)2 at the same time points. ANOVA. (B) In vivo Stx2-neutralizing activity of VHHs. Naïve mice (3–5 mice/group) were injected i.v. with 1LD100 (0.05 pmoles/mouse) of rStx2, and simultaneously injected with 100 μl of PBS, 33 pmoles of 2vb27, 100 pmoles (2vb27)2 or 10 pmoles of (2vb27)2-SA. *p < 0.05 and **p < 0.01, vs 2vb27 and PBS groups. Log-Rank test. (C) Minimal effective dose of (2vb27)2-SA. Naïve mice (5–6 mice/group) were injected i.v. with 1LD100 (0.05 pmoles/mouse) of rStx2 and simultaneously injected with 100 μl of PBS (control) or with 1, 0.1 or 0.05 pmoles of (2vb27)2-SA. Animals were observed three times a day and survival was recorded. **p < 0.005 and ***p < 0.001 vs PBS, Log-Rank test.
Figure 4
Figure 4. In vivo protective capacity of (2vb27)2-SA in the incremental split-dose model of Stx2 intoxication.
One LD100 of rStx2 (0.05 pmoles/mouse) was divided in four consecutive daily doses (the first two doses were 0.009 pmoles/mouse, grey arrows; and the last two doses were 0.016 pmoles/mouse, black arrows). (A) Therapeutic window. Mice (4–6 mice/group) were injected with the split dose model and with (2vb27)2-SA at day 2, or day 3, or day 4. *p < 0.05 vs PBS, Log-Rank test. (B) Lowest protective dose. Mice were injected with the split-dose model and treated with different doses [1 pmoles (n = 3), 0.1 pmoles (n = 6), 0.05 pmoles (n = 15) or 0.01 pmoles (n = 6)] of (2vb27)2-SA on day 3 (dashed arrow). Mice were injected with 100 μl of PBS as control (n = 12). ***p < 0.0001 vs PBS, Log-Rank test. (C,D) Systemic signs of Stx2-associated toxicity. Mice were injected with the split-dose model and treated with 0.1 pmoles of (2vb27)2-SA on day 3. Each time point represents the mean ± SEM for 6–8 mice/group. (C) Relative number of PMN cells. *p < 0.05, **p < 0.01 vs (2vb27)2-SA. Student t test. (D) Plasma urea levels. ***p < 0.001 vs (2vb27)2-SA. Student t test.
Figure 5
Figure 5. In vivo protective capacity of (2vb27)2-SA against STEC-induced pathogenicity.
(A) Survival rates in response to a lethal intragastric (i.g.) challenge with STEC. Seventeen- to nineteen-day-old mice (n = 5–6 mice/group) were injected i.p. with 50 μl of PBS, 0.5 pmoles of (2vb27)2-SA or 0.1 pmoles of (2vb27)2-SA. Immediately after injection, mice were infected i.g. with 4 × 1011 CFU/kg of Stx2-producing E. coli O157:H7. **p < 0.005 vs PBS. Log-Rank test. (B) Renal Stx2-induced toxicity. Plasmatic urea levels at 72 and 96 h post-STEC challenge were measured as a correlate of renal damage. Each time point represents the mean ± SEM of 5–6 mice/group. *p < 0.05 and **p < 0.01 vs PBS at the same time points. ANOVA. (C,D) Systemic signs of Stx2 toxicity. Mice were bled at 72 and 96 h post-STEC challenge, and total and differential counts of leukocytes were assayed. Each time point represents the mean ± SEM from 5–6 mice/group. (C) Relative number of PMN cells. *p < 0.05 vs PBS at the same time points. ANOVA. (D) Total number of leukocytes.
Figure 6
Figure 6. Involvement of the reticuloendothelial system in protection against Stx2 by (2vb27)2-SA.
(A) Depletion of splenic macrophages by Lip-Clod treatment. Adult BALB/c mice (3–4 mice/group) were injected i.v. with a unique dose of 200 μl of Lip-Clod or Lip-PBS. Twenty-four hours later, the animals were injected i.p. with 25 μl (diluted 1:4 in PBS) of a rabbit anti-mouse platelet antiserum. Five hours after antibody inoculation, mice were bled and platelets were counted in a veterinary automatic hematology analyzer. ***p < 0.001. NS: not significant. ANOVA. (B) Protection of (2vb27)2-SA against rStx2 in the absence of splenic macrophages. Adult BALB/c mice (3 mice/group) were i.v. injected with of 200 μl of Lip-Clod or Lip-PBS. Twenty-four hours later, mice were i.v. injected with 1LD100 of rStx2 pre-incubated 1 h at 37 °C with or without 1 pmol/mouse of (2vb27)2-SA. *p < 0.05 vs Lip-PBS Stx2 and Lip-Clod Stx2. Log-Rank Test.
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