Structure and Function Analysis of Therapeutic Monoclonal Antibodies against Dengue Virus Type 2

Abstract

Dengue virus (DENV) is the most prevalent insect-transmitted viral disease in humans globally, and currently no specific therapy or vaccine is available. Protection against DENV and other related flaviviruses is associated with the development of antibodies against the viral envelope (E) protein. Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV type 2 (DENV-2), none have compared simultaneously the inhibitory activity against a genetically diverse range of strains in vitro, the protective capacity in animals, and the localization of epitopes. Here, with the goal of identifying MAbs that can serve as postexposure therapy, we investigated in detail the functional activity of a large panel of new anti-DENV-2 mouse MAbs. Binding sites were mapped by yeast surface display and neutralization escape, cell culture inhibition assays were performed with homologous and heterologous strains, and prophylactic and therapeutic activity was evaluated with two mouse models. Protective MAbs localized to epitopes on the lateral ridge of domain I (DI), the dimer interface, lateral ridge, and fusion loop of DII, and the lateral ridge, C-C loop, and A strand of DIII. Several MAbs inefficiently inhibited at least one DENV-2 strain of a distinct genotype, suggesting that recognition of neutralizing epitopes varies with strain diversity. Moreover, antibody potency generally correlated with a narrowed genotype and serotype specificity. Five MAbs functioned efficiently as postexposure therapy when administered as a single dose, even 3 days after intracranial infection of BALB/c mice. Overall, these studies define the structural and functional complexity of antibodies against DENV-2 with protective potential.Dengue virus (DENV), a member of the Flaviviridae family of RNA viruses, is related to several other human pathogens of global concern, including yellow fever and tick-borne, West Nile, and Japanese encephalitis viruses. DENV infection in humans occurs after Aedes aegypti or Aedes albopictus mosquito inoculation and results in clinical disease, ranging from a febrile illness (dengue fever [DF]) to a life-threatening hemorrhagic and capillary leak syndrome (dengue hemorrhagic fever [DHF]/dengue shock syndrome [DSS]). Globally, there is significant diversity among DENV strains, including four distinct serotypes (DENV type 1 [DENV-1], DENV-2, DENV-3, and DENV-4) that differ at the amino acid level by 25 to 40%. Additional complexity occurs within each serotype, as genotypes vary from one another by up to 3% at the amino acid level (21,49). No approved antiviral treatment is currently available, and several candidate tetravalent vaccines remain in clinical development (reviewed in reference 11). Because of the increased geographic range of its mosquito vectors, urbanization, and international travel, DENV continues to spread worldwide and now causes an estimated 50 to 100 million infections and 250,000 to 500,000 cases of DHF/DSS per year, with 2.5 billion people at risk (68)....

Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination

“…Although the use of DIII alone as an antigen for DENV vaccine development has been proved to induce antibodies against neutralizing E epitopes (Pierson et al, 2008;Poggianella et al, 2015), recent data have demonstrated the presence of broadly neutralizing antibodies directed against epitopes on DI and DII (Sukupolvi-Petty et al, 2010;Wahala et al, 2009;Williams et al, 2012), and on E dimers (envelope dimer epitopes) that are reactive with all four serotypes Rouvinski et al, 2015).…”

“…Since most of neutralizing epitopes on E appear to be conformational in nature (Sukupolvi-Petty et al, 2010), obtaining the E protein in its native conformation is critical for the development of an efficient vaccine against DENV . Several C-terminal truncated versions of E, in which the stem and the transmembrane anchor of the protein have been removed, have been developed for DNA and protein subunit immunizations (Coller et al, 2011;Guzmán et al, 2003;Mani et al, 2013;Ocazionez Jimenez & Lopes da Fonseca, 2000).…”

“…While neutralizing epitopes have been found on all three domains and on the dimer interface Rothman, 2011;Rouvinski et al, 2015;Sukupolvi-Petty et al, 2010), antibodies against the upper lateral surface of DIII are described as the ones with the highest neutralizing capacity (Crill & Roehrig, 2001;Gromowski et al, 2008;Lok et al, 2008;Matsui et al, 2009). We have recently shown that DNA vaccination with DIII-based constructs of all four serotypes results in highly serotype-specific neutralizing responses without induction of antibody-dependent enhancement of infection (Poggianella et al, 2015).…”

Functional analysis of dengue virus (DENV) type 2 envelope protein domain 3 type-specific and DENV complex-reactive critical epitope residues

“…1b). The G330D substitution occurs naturally and is characteristic of the DENV-2 sylvatic genotype (Chambers et al, 1990;Pitcher et al, 2012;Sukupolvi-Petty et al, 2010).…”

Single-Cell Analysis of B Cell/Antibody Cross-Reactivity Using a Novel Multicolor FluoroSpot Assay

“…Therefore, we screened by ELISA a panel of mAbs to identify those with serotype-specific reactivity. Three murine mAbs, all binding E domain III (anti-DENV1 E95, anti-DENV2 E96, and anti-DENV4 E88) (17)(18)(19) and one human E domain I/II-directed mAb (anti-DENV3 5J7) (14,20), were selected. The epitopes targeted are maturation insensitive; thus, the maturation state of the virion should not affect binding of the detection mAbs (20,21).…”