Isolation and Chimerization of a Highly Neutralizing Antibody Conferring Passive Protection against Lethal Bacillus anthracis Infection

Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD50 B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD50 of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax

Evaluating the Synergistic Neutralizing Effect of Anti-Botulinum Oligoclonal Antibody Preparations

<div><p>Botulinum neurotoxins (BoNT) are considered some of the most lethal known substances. There are seven botulinum serotypes, of which types A, B and E cause most human botulism cases. Anti-botulinum polyclonal antibodies (PAbs) are currently used for both detection and treatment of the disease. However, significant improvements in immunoassay specificity and treatment safety may be made using monoclonal antibodies (MAbs). In this study, we present an approach for the simultaneous generation of highly specific and neutralizing MAbs against botulinum serotypes A, B, and E in a single process. The approach relies on immunization of mice with a trivalent mixture of recombinant C-terminal fragment (Hc) of each of the three neurotoxins, followed by a parallel differential robotic hybridoma screening. This strategy enabled the cloning of seven to nine MAbs against each serotype. The majority of the MAbs possessed higher anti-botulinum ELISA titers than anti-botulinum PAbs and had up to five orders of magnitude greater specificity. When tested for their potency in mice, neutralizing MAbs were obtained for all three serotypes and protected against toxin doses of 10 MsLD₅₀–500 MsLD₅₀. A strong synergistic effect of up to 400-fold enhancement in the neutralizing activity was observed when serotype-specific MAbs were combined. Furthermore, the highly protective oligoclonal combinations were as potent as a horse-derived PAb pharmaceutical preparation. Interestingly, MAbs that failed to demonstrate individual neutralizing activity were observed to make a significant contribution to the synergistic effect in the oligoclonal preparation. Together, the trivalent immunization strategy and differential screening approach enabled us to generate highly specific MAbs against each of the A, B, and E BoNTs. These new MAbs may possess diagnostic and therapeutic potential.</p></div

Titer, isotype, and specificity of anti-BoNT B MAbs.

a<p>Ig isotypes (H = heavy chain, L = light chain) were determined using a commercial kit (AbD Serotec, USA).</p>b<p>Titers were measured by ELISA using toxoids as capture antigens.</p>C<p>Specificity was determined by dividing the homologous titer with the heterologous cross-titer. The minimum titer was set at 100.</p

Titer, isotype, and specificity of anti-BoNT A MAbs.

a<p>Ig isotypes (H = heavy chain, L = light chain) were determined using a commercial kit (AbD Serotec, USA).</p>b<p>Titers were measured by ELISA using toxoids as capture antigens.</p>C<p>Specificity was determined by dividing the homologous titer with the heterologous cross-titer. The minimum titer was set at 100.</p

Individual neutralizing activity.

<p>A constant dilution of individual MAb ascitesfluids (1∶100) was pre-incubated with different toxin doses as described in materials and methods and then injected into mice. The results indicate the maximal toxin dose that each MAb could neutralize with respect to its anti-toxin ELISA titer. The MAb was considered neutralizing if 100% survival was achieved.</p

Binding Native toxin.

<p>Anti-native BoNT A, B and E titers of MAbs and control PAbs were determined by s-ELISA. Rabbit anti serotype-specific complex A, B, or E PAbs were used to capture toxin, and donkey anti-mouse IgG HRP-conjugate was used to detect bound antibodies. Titers were determined as the last dilution having signal at 450 nm greater than three standard deviations above control naive sera. Striped bars represent mouse anti-Hc PAb controls.</p

Anti-serotype A, B, and E ELISA titers in mice immunized by a trivalent protocol.

<p><b>A.</b> Immunization scheme <b>-</b> Ten female Balb/c mice were immunized s.c. with a trivalent vaccine containing 5 µg of each of the three Hc fragments, HcA, HcB, and HcE, in CFA (first injection) or IFA (second and third injections). Booster immunizations of trivalent or monovalent (HcE alone) vaccines were given i.m. at 4-week intervals. After three boosters, the immunization regimen was split into two (I and II). Then, two groups of five mice were differentially injected according to their ELISA titers. Mice were bled for titer analysis ten days after each immunization. <b>B</b>. ELISA titer development from week 13 through week 27 for the two combined protocols. As no significant difference in ELISA titer was observed between the two immunization groups (I and II), the data represent the geometric mean of all 10 mice with 95% confidence levels. <b>C</b>. Final titers of anti-serotype A, B, and E (at week 27) in the trivalent combined protocols compared to titers of anti-serotype A in the HcA monovalent control. Geometric means with 95% confidence levels are presented for each of the 10 animal groups (trivalent protocol: anti-A –23,600, anti-B –32,000, anti-E –28,500; monovalent protocol: anti-A –25,900).</p

Neutralizing activity of oligoclonal combinations.

<p>Different toxin doses were pre-incubated with combinations of equally diluted MAb ascites fluids (final dilution 1∶200) and then injected to mice. The results indicate the maximal toxin dose that mice could withstand. Anti-serotype B MAb results are zoomed separately. Anti-serotype A MAb panel: <b><i>Seven</i></b> – [A-4, A-1, A-6, A-2, A-3, A-8, A-7]; <b><i>Four-EP</i></b> – [epitope recognition based MAbs A-4, A-1, A-3, A-8]; <b><i>Four-Neut</i></b> – [neutralizing MAbs A-4, A-1, A-6, A-2]; <b><i>Three</i></b> – [A-4, A-1, A-8 or A-3]. Anti-serotype B MAb panel: <b><i>Seven</i></b> – [B-4, B-2, B-1, B-3, B-6, B-5, B-7]; <b><i>Two (B-1)</i></b> – [B-4, B-1]; <b><i>Two (B-5)</i></b> – [B-4, B-5]. Anti-serotype E MAb panel: <b><i>Eight</i></b> – [E-2, E-3, E-4, E-5, E-6, E-7, E-8, E-1]; <b><i>Six-Neut</i></b> – [neutralizing MAbs E-2, E-3, E-4, E-5, E-7, E-1]; <b><i>Four-EP</i></b> – [epitope recognition based MAbs E-2, E-3, E-8, E-1].</p