Identification of Chimpanzee Fab Fragments by Repertoire Cloning and Production of a Full-Length Humanized Immunoglobulin G1 Antibody That Is Highly Efficient for Neutralization of Dengue Type 4 Virus

Abstract

A safe and effective dengue vaccine is still not available. Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative for the prevention of dengue virus infection. Fab monoclonal antibodies to dengue type 4 virus (DENV-4) were recovered by repertoire cloning of bone marrow mRNAs from an immune chimpanzee and analyzed for antigen binding specificity, V H and V L sequences, and neutralizing activity against DENV-4 in vitro. Fabs 5A7, 3C1, 3E4, and 7G4 were isolated from a library constructed from a chimpanzee following intrahepatic transfection with infectious DENV-4 RNA. Fabs 5H2 and 5D9, which had nearly identical V H sequences but varied in their V L sequences, were recovered from a library constructed from the same chimpanzee after superinfection with a mixture of DENV-1, DENV-2, and DENV-3. In radioimmunoprecipitation, Fab 5A7 precipitated only DENV-4 prM, and Fabs 3E4, 7G4, 5D9, and 5H2 precipitated DENV-4 E but little or no prM. Among the arthropod-borne flaviviruses, the four dengue virus serotypes (dengue type 1 virus [DENV-1], DENV-2, DENV-3, and DENV-4) that constitute a serologically distinct subgroup are most important in terms of human morbidity and geographic distribution. Dengue viruses cause dengue outbreaks and major epidemics in most tropical and subtropical areas where Aedes albopictus and Aedes aegypti mosquitos are abundant. Dengue virus infection produces fever, rash, and joint pain in humans. A more severe and life-threatening form of dengue, characterized by hemorrhagic fever and hemorrhagic shock, has occurred with increasing frequency in Southeast Asia and Central and South America, where all four dengue virus serotypes circulate. The underlying cause of severe dengue remains controversial (23,53). An association of severe dengue with increased viral replication has been reported recently (61). A safe and effective vaccine against dengue is currently not available.The dengue virus contains a positive-strand RNA genome coding for a polyprotein that is cleaved co-and posttranslationally by a combination of cellular and viral proteases to generate the individual viral proteins (9,19,40). Dengue virus prM and E structural proteins and nonstructural NS1 protein are glycosylated. The prM glycoprotein is further cleaved by the cellular enzyme furin following viral assembly, generating M, which is present in the mature virus (58). Flavivirus prM and E form heterodimers, which are assembled into viral particles during infection (62). In this manner, the prM serves to protect the functional integrity of E from acid-induced conformational change (26,31). The E glycoprotein is responsible for cell attachment, possibly mediated by a receptor, and for fusion with the cell membranes following viral entry.Mouse monoclonal antibodies against the dengue viruses have been valuable for dengue virus serotype determination (20,27). Studies in which monoclonal antibodies were used against dengue virus and other flaviviruses have also provided valuable...

Monoclonal antibody-mediated enhancement of dengue virus infectionin vitroandin vivoand strategies for prevention

“…Mutations in the Fc region of IgG 1A5 were generated in the pFab CMV expression vector by overlap extension PCR (15). Briefly, substitutions of C 1348 3 G, G 1356 3 T, and C 1359 3 T according to the numbering system of ref.…”

“…DENV-4 cDNA concentration curves were also generated, and the conversion factor was Ϸ0.034 Eq FFU per copy. Radioimmunoprecipitation was used to semiquantify the anti-NS1 antibodies present in monkey sera 2, 4, and 6 weeks after challenge as an independent measurement of DENV-4 infection as described (15).…”

“…We have identified chimpanzee-human chimeric IgG1 mAbs capable of neutralizing or binding to one or more DENV serotypes (14,15). Cross-reactive IgG 1A5 neutralizes DENV-1 and DENV-2 more efficiently than DENV-3 and DENV-4, and type-specific IgG 5H2 neutralizes DENV-4 at a high titer (14,15).…”

“…Cross-reactive IgG 1A5 neutralizes DENV-1 and DENV-2 more efficiently than DENV-3 and DENV-4, and type-specific IgG 5H2 neutralizes DENV-4 at a high titer (14,15). Analysis of antigenic variants has localized the IgG 1A5 binding site to the conserved fusion peptide in E (11).…”

Construction of Human Fab (Y1/K) Library and Identification of Human Monoclonal Fab Possessing Neutralizing Potency against Japanese Encephalitis Virus

“…4.5 ϫ 10 7 cells using an RNA extraction kit (Stratagene) and mRNA was reverse-transcribed using oligo(dT) as a primer (ThermoScript RT-PCR System, Invitrogen). The full-length human Fab DNA product was created by PCR as described elsewhere (3,23). Briefly, the κ light-chain DNA was amplified from the cDNA product by PCR with seven pairs of human κ light-chain familyspecific 5' primers and a 3' primer in the constant domain.…”