An Engineered N-Glycosylated Dengue Envelope Protein Domain III Facilitates Epitope-Directed Selection of Potently Neutralizing and Minimally Enhancing Antibodies.

The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the target of multiple potently neutralizing antibodies. One such antibody is 3H5, a mouse antibody that binds strongly to EDIII and potently neutralizes DENV serotype 2 (DENV-2) with unusually minimal antibody-dependent enhancement (ADE). To selectively display the binding epitope of 3H5, we strategically modified DENV-2 EDIII by shielding other known epitopes with engineered N-glycosylation sites. The modifications resulted in a glycosylated EDIII antigen termed "EDIII mutant N". This antigen was successfully used to sift through a dengue-immune scFv-phage library to select for scFv antibodies that bind to or closely surround the 3H5 epitope. The selected scFv antibodies were expressed as full-length human antibodies and showed potent neutralization activity to DENV-2 with low or negligible ADE resembling 3H5. These findings not only demonstrate the capability of the N-glycosylated EDIII mutant N as a tool to drive an epitope-directed antibody selection campaign but also highlight its potential as a dengue immunogen. This glycosylated antigen shows promise in focusing the antibody response toward a potently neutralizing epitope while reducing the risk of antibody-dependent enhancement.

Blockade-of-Binding Activities toward Envelope-Associated, Type-Specific Epitopes as a Correlative Marker for Dengue Virus-Neutralizing Antibody.

Humans infected with dengue virus (DENV) acquire long-term protection against the infecting serotype, whereas cross-protection against other serotypes is short-lived. Long-term protection induced by low levels of type-specific neutralizing antibodies can be assessed using the virus-neutralizing antibody test. However, this test is laborious and time-consuming. In this study, a blockade-of-binding enzyme-linked immunoassay was developed to assess antibody activity by using a set of neutralizing anti-E monoclonal antibodies and blood samples from dengue virus-infected or -immunized macaques. Diluted blood samples were incubated with plate-bound dengue virus particles before the addition of an enzyme-conjugated antibody specific to the epitope of interest. Based on blocking reference curves constructed using autologous purified antibodies, sample blocking activity was determined as the relative concentration of unconjugated antibody that resulted in the same percent signal reduction. In separate DENV-1-, -2-, -3-, and -4-related sets of samples, moderate to strong correlations of the blocking activity with neutralizing antibody titers were found with the four type-specific antibodies 1F4, 3H5, 8A1, and 5H2, respectively. Significant correlations were observed for single samples taken 1 month after infection as well as samples drawn before and at various time points after infection/immunization. Similar testing using a cross-reactive EDE-1 antibody revealed a moderate correlation between the blocking activity and the neutralizing antibody titer only for the DENV-2-related set. The potential usefulness of the blockade-of-binding activity as a correlative marker of neutralizing antibodies against dengue viruses needs to be validated in humans. IMPORTANCE This study describes a blockade-of-binding assay for the determination of antibodies that recognize a selected set of serotype-specific or group-reactive epitopes in the envelope of dengue virus. By employing blood samples collected from dengue virus-infected or -immunized macaques, moderate to strong correlations of the epitope-blocking activities with the virus-neutralizing antibody titers were observed with serotype-specific blocking activities for each of the four dengue serotypes. This simple, rapid, and less laborious method should be useful for the evaluation of antibody responses to dengue virus infection and may serve as, or be a component of, an in vitro correlate of protection against dengue in the future.

Differential critical residues on the overlapped region of the non-structural protein-1 recognized by flavivirus and dengue virus cross-reactive monoclonal antibodies.

The non-structural protein-1 (NS1) of dengue virus (DENV) contributes to several functions related to dengue disease pathogenesis as well as diagnostic applications. Antibodies against DENV NS1 can cross-react with other co-circulating flaviviruses, which may lead to incorrect diagnosis. Herein, five anti-DENV NS1 monoclonal antibodies (mAbs) were investigated. Four of them (1F11, 2E3, 1B2, and 4D2) cross-react with NS1 of all four DENV serotypes (pan-DENV mAbs), whereas the other (2E11) also reacts with NS1 of other flaviviruses (flavi-cross-reactive mAb). The binding epitopes recognized by these mAbs were found to overlap a region located on the disordered loop of the NS1 wing domain (amino acid residues 104 to 123). Fine epitope mapping employing phage display technology and alanine-substituted DENV2 NS1 mutants indicates the critical binding residues W115, K116, and K120 for the 2E11 mAb, which are conserved among flaviviruses. In contrast, the critical binding residues of four pan-DENV mAbs include both flavi-conserved residues (W115 to G119) and DENV-conserved flanking residues (K112, Y113, S114 and A121, K122). Our results highlight DENV-conserved residues in cross-reactive epitopes that distinguish pan-DENV antibodies from the flavi-cross-reactive antibody. These antibodies can be potentially applied to differential diagnosis of DENV from other flavivirus infections.

A replication competent luciferase-secreting DENV2 reporter for sero-epidemiological surveillance of neutralizing and enhancing antibodies.

Dengue virus (DENV) specific neutralizing and enhancing antibodies play crucial roles in dengue disease prevention and pathogenesis. DENV reporters are gaining popularity in the evaluation of these antibodies; their accessibility and acceptance may improve with more efficient production systems and indications of their antigenic equivalence to the wild-type virus. This study aimed to generate a replication competent luciferase-secreting DENV reporter (LucDENV2) and evaluate its feasibility in neutralizing and infection-enhancing antibody assays in comparison with wild-type DENV2, strain 16681, and a luciferase-secreting, single-round infectious DENV2 reporter (LucSIP). LucDENV2 replicated to similarly high levels as that of the parent 16681 virus in a commonly used mosquito cell line. LucDENV2 was neutralized in an antibody concentration-dependent manner by a monoclonal antibody specific to the flavivirus fusion loop and two antibodies specific to the E domain III, which closely resembled the neutralization patterns employing the LucSIP and wild-type DENV2. Parallel analysis of LucDENV2 and wild-type DENV2 revealed good agreement between the luciferase-based and focus-based neutralization and enhancement assays in a 96-well microplate format when employed against a set of clinical sera, suggesting comparable antigenic properties of LucDENV2 with those of the parent virus. The high-titer, replication competent, luciferase-secreting DENV reporter presented here should be a useful tool for fast and reliable quantitation of neutralizing and infection-enhancing antibodies in populations living in DENV-endemic areas.

Cross-reactive antibodies targeting surface-exposed non-structural protein 1 (NS1) of dengue virus-infected cells recognize epitopes on the spaghetti loop of the ß-ladder domain.

Non-structural protein 1 (NS1) is a glycoprotein component of dengue virus (DENV) that is essential for viral replication, infection and immune evasion. Immunization with NS1 has been shown to elicit antibody-mediated immune responses which protect mice against DENV infections. Here, we obtained peripheral blood mononuclear cells from human subjects with secondary dengue infections, which were used to construct a dengue immune phage library displaying single-chain variable fragments. Phage selective for DENV NS1 were obtained by biopanning. Twenty-one monoclonal antibodies (mAbs) against DENV NS1 were generated from the selected phage and characterized in detail. We found most anti-NS1 mAbs used IGHV1 heavy chain antibody genes. The mAbs were classified into strongly and weakly-reactive groups based on their binding to NS1 expressed in dengue virus 2 (DENV2)-infected cells. Antibody binding experiments with recombinant NS1 proteins revealed that the mAbs recognize conformational epitopes on the ß-ladder domain (amino acid residues 178-273) of DENV NS1. Epitope mapping studies on alanine-substituted NS1 proteins identified distinct but overlapping epitopes. Protruding amino acids distributed around the spaghetti loop are required for the binding of the strongly-reactive mAbs, whereas the recognition residues of the weakly-reactive mAbs are likely to be located in inaccessible sites facing toward the cell membrane. This information could guide the design of an NS1 epitope-based vaccine that targets cross-reactive conserved epitopes on cell surface-associated DENV NS1.

Dengue and the Lectin Pathway of the Complement System.

Dengue is a mosquito-borne viral disease causing significant health and economic burdens globally. The dengue virus (DENV) comprises four serotypes (DENV1-4). Usually, the primary infection is asymptomatic or causes mild dengue fever (DF), while secondary infections with a different serotype increase the risk of severe dengue disease (dengue hemorrhagic fever, DHF). Complement system activation induces inflammation and tissue injury, contributing to disease pathogenesis. However, in asymptomatic or primary infections, protective immunity largely results from the complement system's lectin pathway (LP), which is activated through foreign glycan recognition. Differences in N-glycans displayed on the DENV envelope membrane influence the lectin pattern recognition receptor (PRR) binding efficiency. The important PRR, mannan binding lectin (MBL), mediates DENV neutralization through (1) a complement activation-independent mechanism via direct MBL glycan recognition, thereby inhibiting DENV attachment to host target cells, or (2) a complement activation-dependent mechanism following the attachment of complement opsonins C3b and C4b to virion surfaces. The serum concentrations of lectin PRRs and their polymorphisms influence these LP activities. Conversely, to escape the LP attack and enhance the infectivity, DENV utilizes the secreted form of nonstructural protein 1 (sNS1) to counteract the MBL effects, thereby increasing viral survival and dissemination.

A simple approach to identify functional antibody variable genes in murine hybridoma cells that coexpress aberrant kappa light transcripts by restriction enzyme digestion.

BACKGROUND: Specific binding to target protein epitopes by a mouse monoclonal antibody (mAb) relies on its variable domains. However, the isolation of functional variable gene transcripts is sometimes hindered by co-expression of aberrant transcripts in hybridoma cells. OBJECTIVE: To develop general strategies for identifying the functional variable transcripts of both heavy (VH) and kappa light (Vκ) chains from mouse hybridomas. METHODS: VH and Vκ genes of anti-dengue hybridoma clones were PCR-amplified using set of degenerate primers covering all mouse immunoglobulin families. Vκ amplicons were additionally digested with BciVI to eliminate aberrant Vκ transcripts. The productive VH and Vκ sequences were identified by Immunogenetics (IMGT) database analysis and cloned into a dual human IgG expression vector to generate chimeric antibodies (chAbs) in mammalian cells. The reactivity of chAbs was tested by immunoblot and immunofluorescent assays. RESULTS: Among 17 tested hybridoma clones, 400 bp Vκ amplicons were obtained using eight different Vκ primers. Amplicons from productive Vκ transcripts are resistant to BciVI digestion, whereas BciVI-digested amplicons indicated aberrant Vκ transcripts. 500-bp productive VH amplicons could be obtained from all clones using a set of five VH primers. The productive VH/Vκ genes of three anti-dengue NS1 mAbs (m2G6, m1F11 and m1A4) were cloned and mouse-human chAbs were generated. The binding reactivities of the chAbs to dengue NS1 were similar to the original mAbs. CONCLUSIONS: A general protocol to identify productive/functional VH and Vκ genes was demonstrated. The method is useful for producing chAbs and genetic archiving of valuable hybridoma cell lines.

Antiviral immune responses in H5N1-infected human lung tissue and possible mechanisms underlying the hyperproduction of interferon-inducible protein IP-10.

Information on the immune response against H5N1 within the lung is lacking. Here we describe the sustained antiviral immune responses, as indicated by the expression of MxA protein and IFN-alpha mRNA, in autopsy lung tissue from an H5N1-infected patient. H5N1 infection of primary bronchial/tracheal epithelial cells and lung microvascular endothelial cells induced IP-10, and also up-regulated the retinoic acid-inducible gene-I (RIG-I). Down-regulation of RIG-I gene expression decreased IP-10 response. Co-culturing of H5N1-infected pulmonary cells with TNF-alpha led to synergistically enhanced production of IP-10. In the absence of viral infection, TNF-alpha and IFN-alpha also synergistically enhanced IP-10 response. Methylprednisolone showed only a partial inhibitory effect on this chemokine response. Our findings strongly suggest that both the H5N1 virus and the locally produced antiviral cytokines; IFN-alpha and TNF-alpha may have an important role in inducing IP-10 hyperresponse, leading to inflammatory damage in infected lung.