Development of Therapeutic Monoclonal Antibodies for Emerging Arbovirus Infections.

Antibody-based passive immunotherapy has been used effectively in the treatment and prophylaxis of infectious diseases. Outbreaks of emerging viral infections from arthropod-borne viruses (arboviruses) represent a global public health problem due to their rapid spread, urging measures and the treatment of infected individuals to combat them. Preparedness in advances in developing antivirals and relevant epidemiological studies protect us from damage and losses. Immunotherapy based on monoclonal antibodies (mAbs) has been shown to be very specific in combating infectious diseases and various other illnesses. Recent advances in mAb discovery techniques have allowed the development and approval of a wide number of therapeutic mAbs. This review focuses on the technological approaches available to select neutralizing mAbs for emerging arbovirus infections and the next-generation strategies to obtain highly effective and potent mAbs. The characteristics of mAbs developed as prophylactic and therapeutic antiviral agents for dengue, Zika, chikungunya, West Nile and tick-borne encephalitis virus are presented, as well as the protective effect demonstrated in animal model studies.

A natural carrier effect and the generation of specific antibodies to biologically active peptides.

Production of specific antibodies to haptens, especially antipeptides, without interference by carrier protein, is desirable. The bradykinin-potentiating peptides (BPPs) are a family of pyroglutamyl proline-rich oligopeptides with strong antihypertensive properties. In this work, the production of antibodies to BPPs by use of an efficient immunization protocol in mice genetically modified for the high antibody responsiveness (H(III) line) is described. Although it was possible to induce antibody production by single-dose administration of free BPPs, higher antibody titers were obtained in mice preimmunized with carrier protein before administration of peptides conjugated to this carrier. Interestingly, both mouse groups had a higher titer of IgG(1) than IgG(2a) isotypes, regardless of prior immunization with the carrier protein. However, a lower titer of IgG(2a) was observed in unprimed mice. A single band of about 27kDa corresponding to the BPP precursor protein was recognized by these antibodies in the cytosol of the Bothrops jararaca venom gland. This work proposes an efficient immunization protocol based on classic studies described for the hapten-carrier effect for generating specific antibodies against biologically active peptides.

A natural carrier effect and the generation of specific antibodies to biologically active peptides

Production of specific antibodies to haptens, especially antipeptides, without interference by carrier protein, is desirable. The bradykinin-potentiating peptides (BPPs) are a family of pyroglutamyl proline-rich oligopeptides with strong antihypertensive properties. In this work, the production of antibodies to BPPs by use of an efficient immunization protocol in mice genetically modified for the high antibody responsiveness (HIII line) is described. Although it was possible to induce antibody production by single-dose administration of free BPPs, higher antibody titers were obtained in mice preimmunized with carrier protein before administration of peptides conjugated to this carrier. Interestingly, both mouse groups had a higher titer of IgG1 than IgG2a isotypes, regardless of prior immunization with the carrier protein. However, a lower titer of IgG2a was observed in unprimed mice. A single band of about 27 kDa corresponding to the BPP precursor protein was recognized by these antibodies in the cytosol of the Bothrops jararaca venom gland. This work proposes an efficient immunization protocol based on classic studies described for the hapten-carrier effect for generating specific antibodies against biologically active peptides.

Design, production, and characterization of recombinant neocarzinostatin apoprotein in Escherichia coli.

Neocarzinostatin (NCS) is the first discovered anti-tumor antibiotic having an enediyne-containing chromophore and an apoprotein with a 1:1 complex. An artificial gene library for NCS apoprotein (apo-NCS) production in Escherichia coli was designed and constructed on a phage-display vector, pJuFo. The recombinant phages expressing pre-apo-NCS protein were enriched with a mouse anti-apo-NCS monoclonal antibody, 1C7D4. The apo-NCS gene (encsA) for E. coli was successfully cloned, and then re-cloned into the pRSET A vector. After the his-tagged apo-NCS protein had been purified and cleaved with enterokinase, the binding properties of the recombinant protein as to ethidium bromide (EtBr) were studied by monitoring of total fluorescence intensity and fluorescence polarization with a BEACON 2000 system and GraphPad Prism software. A dissociation constant of 4.4 +/- 0.3 microM was obtained for recombinant apo-NCS in the fluorescence polarization study. This suggests that fluorescence polarization monitoring with EtBr as a chromophore mimic may be a simplified method for the characterization of recombinant apo-NCS binding to the NCS chromophore. When Phe78 on apo-NCS was substituted with Trp78 by site-directed mutagenesis using a two stage megaprimer polymerase chain reaction, the association of the apo-NCS mutant and EtBr observed on fluorescence polarization analysis was of the same degree as in the case of the wild type, although the calculated maximum change (DeltaIT(max)) in total fluorescence intensity decreased from 113.9 to 31.3. It was suggested that an environmental change of the bound EtBr molecule on F78W might have dramatically occurred as compared with in the case of wild type apo-NCS. This combination of monitoring of fluorescence polarization and total fluorescence intensity will be applicable for determination and prediction of the ligand state bound or associated with the target protein. The histone-specific proteolytic activity was also re-investigated using this recombinant apo-NCS preparation, and calf thymus histone H1, H2A, H2B, H3, and H4. The recombinant apo-NCS does not act as a histone protease because a noticeable difference was not observed between the incubation mixtures with and without apo-NCS under our experimental conditions.

Interactions between cationic liposomes and an antigenic protein The physical chemistry of the immunoadjuvant action

The 18 kDa antigenic protein from Mycobacterium leprae (P) or its N- acyl derivative (AP) was incorporated in dioctadecyldimethylammonium bromide (DODAB) liposomes in water or in phosphate-buffered saline (PBS). In water, 100% P incorporation in liposomes contrasts with 65% in PBS. There is 75-80% AP incorporation to liposomes in water against 55-65% in PBS, showing that attachment of hydrophobic residues to the protein, instead of increasing, further decreases incorporation to the liposomes. From protein adsorption on latex, P affinity is larger than AP affinity for the latex surface whereas limiting adsorption for AP is much larger than that obtained for P, possibly due to AP aggregation in solution. P-induced rupture of liposomes containing [14C]sucrose was evaluated from dialysis of protein/liposomes mixtures. In water, P incorporation to the liposomes causes leakage of radioactive contents contrasting with the absence of leakage for P incorporation in PBS. Immunization tests for delayed type hypersensitivity indicate a enhancement of cell-mediated immunological response towards P/DODAB complexes that is not obtained for the isolated protein. Absence of leakage for P in PBS is associated with a P 'lying-over' on the liposome and optimization of protein presentation to the immunological system.