A Naturally Occurring Antibody Fragment Neutralizes Infectivity of Diverse Infectious Agents

Abstract

A phosphorylated peptide, named K40H, derived from the constant region of IgMs was detected in human serum by liquid chromatography coupled to high-resolution mass spectrometry. Synthetic K40H proved to exert a potent in vitro activity against fungal pathogens, and to inhibit HIV-1 replication in vitro and ex vivo. It also showed a therapeutic effect against an experimental infection by Candida albicans in the invertebrate model Galleria mellonella. K40H represents the proof of concept of the innate role that naturally occurring antibody fragments may exert against infectious agents, shedding a new light upon the posthumous role of antibodies and opening a new scenario on the multifaceted functionality of humoral immunity.

Figure 1. Time kinetics of K40H killing of Candida albicans.

Viable yeast cells were incubated up to 6 h in the absence (control) or presence of K40H at different concentrations. At different times (30, 60, 120, 240, and 360 min), the yeast suspensions were plated on Sabouraud dextrose agar, and colony forming units were enumerated after 48 h. Percentual killing was calculated with reference to the number of colonies in controls. Results are representative of at least 2 independent experiments. Each experiment was performed in triplicate.

Figure 2. In vitro and ex vivo activity of K40H against HIV-1.

(A) K40H (2 μM) was added to PBMCs cultures before (pre-) or after (post-) infection with BaL (R5, left panel) and IIIB (X4, right panel) HIV-1. (B) PBMCs from five HIV-1-infected patients (PZ1-5) were cultured in presence of K40H (2 μM). Virus production was assayed in the supernatants by detecting p24 HIV-1 antigen, on day 8 and 12 after infection. Ag production in untreated cultures corresponded to 100% of virus production. For all assay conditions, results are representative of mean values from 5 independent experiments, in all cases variability was less than 10%.

Figure 3. In vivo therapeutic activity of K40H.

Effect of treatment with a single dose of peptide (10 μl from a 86.5 μM solution, 1 h postchallenge) on the survival of Galleria mellonella larvae (16/group) infected with 5 × 10⁵ Candida albicans cells. The survival curve of K40H-treated animals was significantly different (p < 0.0001) from that of control larvae as assessed by the log-rank (Mantel-Cox) test. Data reported are from one representative experiment out of two experiments with comparable results.

Figure 4. Transmission electron microscopy of Candida albicans cells treated with peptide K40H.

Approximately 10⁷ yeast cells were incubated without (panels a–d) or with (panels e–m) K40H (32.5 μM) for 1 h. Treated cells presented membrane retraction and extensive cytoplasm disintegration (e,m), vacuolation (g), perinuclear and granular nuclear alterations (f,h–l), swelling and separation of an outer layer of the cell wall (j,k).

Figure 5. Scanning electron microscopy of Candida albicans cells treated with peptide K40H.

Approximately 10⁷ yeast cells were incubated without (panels A,B) or with (panels C–F) K40H (130 μM) for 3 h. Treated cells presented a rugged surface and detachment of an outer layer of the yeast cell wall. Masses of cellular debris were seen.

Figure 6. Binding of biotin-labeled K40H to F-actin on Candida albicans.

Yeast cells were treated with biotin-labeled K40H (130 μM) for 3 h, fixed with formaldehyde and permeabilised with Triton X-100. Peptide-untreated cells (A) and peptide-treated cells (B) were stained with DAPI, streptavidin-FITC, and phalloidin-rodamine. Panel a: differential interference contrast microscopy; panel b: DAPI; panel c: streptavidin FITC; panel d: phalloidin-rhodamine; panel e: merge with superposition of blue, red and green fluorescence outputs (b–d) of the same cells; panel f: co-localisation points of c and d. Biotin-labeled peptide co-localises with F-actin. Scale bar, 20 μm.

Figure 7. Actin polymerisation by K40H.

Polymerisation of pyrene-labeled monomeric actin (G-actin) was assayed with K40H at 200 μM in comparison with a positively reacting peptide C7H2, in a time dependent kinetics. The fluorescence at 410 nm was measured (RFU, relative fluorescent units). At the end of 50 min of the stabilized pyrene-actin (NC, negative control), ATP-containing polymerisation buffer was added (arrow) and the steep polymerisation and intensity (PC, positive control) was compared to that obtained with the peptides.