Generation of neutralizing human monoclonal antibodies against parvovirus B19 proteins

Abstract

Infections caused by human parvovirus B19 are known to be controlled mainly by neutralizing antibodies. To analyze the immune reaction against parvovirus B19 proteins, four cell lines secreting human immunoglobulin G monoclonal antibodies (MAbs) were generated from two healthy donors and one human immunodeficiency virus type 1-seropositive individual with high serum titers against parvovirus. One MAb is specific for nonstructural protein NS1 (MAb 1424), two MAbs are specific for the unique region of minor capsid protein VP1 (MAbs 1418-1 and 1418-16), and one MAb is directed to major capsid protein VP2 (MAb 860-55D). Two MAbs, 1418-1 and 1418-16, which were generated from the same individual have identity in the cDNA sequences encoding the variable domains, with the exception of four base pairs resulting in only one amino acid change in the light chain. The NS1- and VP1-specific MAbs interact with linear epitopes, whereas the recognized epitope in VP2 is conformational. The MAbs specific for the structural proteins display strong virus-neutralizing activity. The VP1- and VP2-specific MAbs have the capacity to neutralize 50% of infectious parvovirus B19 in vitro at 0.08 and 0.73 microgram/ml, respectively, demonstrating the importance of such antibodies in the clearance of B19 viremia. The NS1-specific MAb mediated weak neutralizing activity and required 47.7 micrograms/ml for 50% neutralization. The human MAbs with potent neutralizing activity could be used for immunotherapy of chronically B19 virus-infected individuals and acutely infected pregnant women. Furthermore, the knowledge gained regarding epitopes which induce strongly neutralizing antibodies may be important for vaccine development.

FIG. 1

Mapping of the epitope recognized by MAb 1424 with fragments derived from the NS1 protein. Fragments of the NS1 protein were produced in E. coli, purified, and tested in an ELISA for the ability to bind MAb 1424. (A) Schematic representation of overlapping protein fragments NS/A to NS/D. Fragments recognized by the NS1-specific MAb are in black. (B) Reactivity of MAb 1424 with different NS1 subfragments tested by ELISA. Only fragment NS/D was strongly recognized by MAb 1424. OD₄₀₅, optical density at 405 nm.

FIG. 2

Fine mapping of the epitope recognized by MAb 1424 with synthetic peptides. Shown are the reactivities of MAb 1424 with overlapping synthetic peptides covering the NS/D protein fragment in an ELISA. The numbers represent amino acid positions in the sequence of the NS1 protein. One peptide (amino acids 511 to 528) was strongly recognized by MAb 1424. OD₄₀₅, optical density at 405 nm.

FIG. 3

Mapping of the epitope recognized by MAbs 1418-1 and 1418-16 by using VP1 protein fragments. Fragments of the VP1 protein were produced in E. coli, bound on ELISA plates precoated with antibodies against a polyhistidine stretch without prior purification, and tested in an ELISA for the ability to bind MAbs 1418-1 and 1418-16. (A) Schematic representation of overlapping protein fragments VP/A to VP/C. Fragments recognized by the VP1-specific MAbs are in black. (B) Reactivity of MAbs 1418-1 (grey columns) and 1418-16 (black columns) with different VP1 subfragments tested by ELISA. Only fragment VP/A was strongly recognized by both MAbs. OD₄₀₅, optical density at 405 nm.

FIG. 4

Capacities of human MAbs to protect erythroid progenitor cells from the cytotoxic effects of infectious parvovirus B19. Neutralization was studied in the CFU-E-derived colony inhibition assay. The results of one of four consistent experiments are illustrated. Means of duplicate determinations of erythroid-cell colonies are shown. In this experiment, the average number of colonies formed in the absence of virus and antibody was 450 per well.