Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin.

Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with high affinity (136 pM) and the BoNT/A2 subtype with low affinity (109 nM), we increased its affinity for BoNT/A2 1,250-fold, to 87 pM, while maintaining high-affinity binding to BoNT/A1 (115 pM). To find the molecular basis for improved cross-reactivity, we determined the X-ray co-crystal structures of wild-type and cross-reactive antibodies complexed to BoNT/A1 at resolutions up to 2.6 A, and measured the thermodynamic contribution of BoNT/A1 and A2 amino acids to wild-type and cross-reactive antibody binding. The results show how an antibody can be engineered to bind two different antigens despite structural differences in the antigen-antibody interface and may provide a general strategy for tuning antibody specificity and cross-reactivity.

A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes.

Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (KD). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had KD values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.

HIV type 1 infection alters cytokine mRNA expression in thymus.

We and others have previously shown that HIV infection of human thymus/liver implants in severe combined immunodeficient (SCID-hu Thy/Liv) mice results in a loss of CD4(+) thymocytes and a decreased recovery of human myeloid and erythroid colony-forming activity. Furthermore, our previous studies have shown that this decrease in colony-forming potential is due to indirect effects, rather than to massive loss of CD34(+) hematopoietic progenitor cells, suggesting that HIV infection might alter expression of hematopoietic cytokines. Herein we have investigated potential HIV-1-induced perturbations of hematolymphoid microenvironments by determining the effect of HIV-1 infection on levels of cytokine mRNAs in human stroma and thymocytes, using the reverse transcription-polymerase chain reaction (RT-PCR). The levels of interleukin 6 (IL-6), interferon gamma (IFN-gamma), and IL-2 RNAs increased and macrophage inflammatory protein 1beta (MIP-1beta) RNA decreased significantly in infected thymocytes. IL-6 RNA levels in stroma also increased somewhat with infection; however, expression of stromal cell-derived factor 1 (SDF-1) by stromal elements was not affected. IL-4 RNA levels were unaffected by infection in both stroma and thymocytes. Antiretroviral drug treatment of the infected animals, which results in restoration of colony-forming potential, tends to restore the cytokine mRNA levels in HIV-1-infected implants to those of mock-infected implants. These results indicate that HIV-1 infection can greatly distort the cytokine profiles in Thy/Liv implants, and suggest that cytokine perturbation could be involved in virus-induced inhibition of hematopoiesis.