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Journal of Virology, December 2004, p. 12910-12918, Vol. 78, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.23.12910-12918.2004

Chimpanzee Fab Fragments and a Derived Humanized Immunoglobulin G1 Antibody That Efficiently Cross-Neutralize Dengue Type 1 and Type 2 Viruses

Ana P. Goncalvez,1 Ruhe Men,1 Claire Wernly,1 Robert H. Purcell,2 and Ching-Juh Lai1*

Molecular Viral Biology Section,1 Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland2

Received 18 May 2004/ Accepted 30 July 2004

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 (PRNT50) titer, ranging from 0.26 to 1.33 µg/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 µg/ml, respectively. IgG1 1A5 also neutralized DENV-3, DENV-4, and the West Nile virus at a PRNT50 titer of approximately 3.2 to 4.2 µg/ml. This humanized antibody represents an attractive candidate for further development of immunoprophylaxis against DENV and perhaps other flavivirus-associated diseases.

* Corresponding author. Mailing address: Molecular Viral Biology Section, Laboratory of Infectious Diseases, NIAID, NIH, Building 50, Room 6349, 50 South Dr., MSC 8009, Bethesda, MD 20892. Phone: (301) 594-2422. Fax: (301) 402-6413. E-mail: clai{at}niaid.nih.gov.

Journal of Virology, December 2004, p. 12910-12918, Vol. 78, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.23.12910-12918.2004




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