Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses - PubMed (original) (raw)

Chimpanzee Fab fragments and a derived humanized immunoglobulin G1 antibody that efficiently cross-neutralize dengue type 1 and type 2 viruses

Ana P Goncalvez et al. J Virol. 2004 Dec.

Abstract

Passive immunization with monoclonal antibodies from humans or nonhuman primates represents an attractive alternative to vaccines for prevention of illness caused by dengue viruses (DENV) and other flaviviruses, including the West Nile virus. In a previous study, repertoire cloning to recover Fab fragments from bone marrow mRNA of chimpanzees infected with all four DENV serotypes (dengue virus serotype 1 [DENV-1] to DENV-4) was described. In that study, a humanized immunoglobulin G1 (IgG1) antibody that efficiently neutralized DENV-4 was recovered and characterized. In this study, the phage library constructed from the chimpanzees was used to recover Fab antibodies against the other three DENV serotypes. Serotype-specific neutralizing Fabs were not identified. Instead, we recovered DENV-neutralizing Fabs that specifically precipitated the envelope protein and were cross-reactive with all four DENV serotypes. Three of the Fabs competed with each other for binding to DENV-1 and DENV-2, although each of these Fabs contained a distinct complementarity determining region 3 (CDR3)-H sequence. Fabs that shared an identical or nearly identical CDR3-H sequences cross-neutralized DENV-1 and DENV-2 at a similar high 50% plaque reduction neutralization test (PRNT(50)) titer, ranging from 0.26 to 1.33 microg/ml, and neutralized DENV-3 and DENV-4 but at a titer 10- to 20-fold lower. One of these Fabs, 1A5, also neutralized the West Nile virus most efficiently among other flaviviruses tested. Fab 1A5 was converted to a full-length antibody in combination with human sequences for production in mammalian CHO cells. Humanized IgG1 1A5 proved to be as efficient as Fab 1A5 for cross-neutralization of DENV-1 and DENV-2 at a titer of 0.48 and 0.95 microg/ml, respectively. IgG1 1A5 also neutralized DENV-3, DENV-4, and the West Nile virus at a PRNT(50) titer of approximately 3.2 to 4.2 microg/ml. This humanized antibody represents an attractive candidate for further development of immunoprophylaxis against DENV and perhaps other flavivirus-associated diseases.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Amino acid sequences of Fabs. (A) sequences of the VL κ light-chain segments; (B) sequences of the VH γ1 heavy-chain segments. FR, framework region. Dashes represent amino acid deletions; and identical amino acids are indicated by dots. The sequence of Fab 3E4 described previously (26) was included for comparison with that of Fab 1A10.

FIG. 2.

FIG. 2.

Analysis of antigen specificity by radioimmunoprecipitation. Radioactive 35S-methionine-labeled lysates separately prepared from Vero cells infected with each of the DENV serotypes (D1 to D4) were used for immune precipitation with Fab 1A5 or Fab 1A10. Lane M, molecular mass markers in kilodaltons. Each of the Fabs precipitated the E protein of each of four DENV serotypes. Note that the E protein often migrated as a doublet or a broad band, probably resulting from differences in glycosylation.

FIG. 3.

FIG. 3.

Analysis of Fab binding to DENV-1 or DENV-2. Fabs 1A5, 1B2, and 1A10 were affinity purified, biotinylated, and used for analysis of binding activity to DENV-1 or DENV-2 by competition ELISA in the presence of competing, unlabeled Fabs. Chimpanzee Fab 1F2, which did not react with any of the DENVs, was used as a negative control. The numbers on the y axes are optical density readings, and the x coordinates represent reciprocal dilutions of the competing Fabs. At the top of each panel, D1 or D2 indicates whether DENV-1 or DENV-2 was used. The insert inside panel A shows the symbol for each Fab; the symbols are the same for all six panels.

FIG. 4.

FIG. 4.

Binding of Fab 1A5 to DENVs and other flaviviruses as measured by Western blotting. Approximately 105 PFU of each virus was applied and separated by polyacrylamide gel electrophoresis. Lanes: D1, DENV-1 strain Hawaii; D2, DENV-2 strain New Guinea B; D3, DENV-3 strain H87; D4, DENV-4 strain 814669; WN/D4, WNV/DENV-4 chimera; JE, JEV strain SA14-14-2; LGT, LGTV strain TP 21. The position of the E protein is indicated. Molecular mass markers are shown on the left, in kilodaltons.

FIG. 5.

FIG. 5.

In vitro neutralization of DENVs and other flaviviruses by humanized IgG1 1A5. The neutralizing activity of IgG1 1A5 against DENV-1 strain Hawaii, DENV-2 strain New Guinea B, DENV-3 strain H87, DENV-4 strain 814669, JEV vaccine strain SA14-14-2, LGTV strain TP 21, and the WNV/DENV-4 chimera was analyzed by a PRNT.

References

    1. Allen, J. M., and B. Seed. 1989. Isolation and expression of functional high-affinity Fc receptor complementary DNAs. Science 243:378-381. - PubMed
    1. Barbas, C. F., III, A. S. Kang, R. A. Lerner, and S. J. Benkovic. 1991. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. USA 88:7978-7982. - PMC - PubMed
    1. Berman, J. E., S. J. Mellis, R. Pollock, C. L. Smith, H. Suh, B. Heinke, C. Kowal, U. Surti, L. Chess, C. R. Cantor, and F. W. Alt. 1988. Content and organization of the human Ig VH locus: definition of three new VH families and linkage to the Ig CH locus. EMBO J. 7:727-738. - PMC - PubMed
    1. Chappel, M. S., D. E. Isenman, M. Everett, Y. Y. Xu, K. J. Dorrington, and M. H. Klein. 1991. Identification of the Fc gamma receptor class I binding site in human IgG through the use of recombinant IgG1/IgG2 hybrid and point-mutated antibodies. Proc. Natl. Acad. Sci. USA 88:9036-9040. - PMC - PubMed
    1. Chothia, C., A. M. Lesk, E. Gherardi, I. M. Tomlinson, G. Walter, J. D. Marks, M. B. Llewelyn, and G. Winter. 1992. Structural repertoire of the human VH segments. J. Mol. Biol. 227:799-817. - PubMed

MeSH terms

Substances

LinkOut - more resources