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.

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Type- and Subcomplex-Specific Neutralizing Antibodies against Domain III of Dengue Virus Type 2 Envelope Protein Recognize Adjacent Epitopes

Journal of Virology ◽

10.1128/jvi.00432-07 ◽

2007 ◽

Vol 81 (23) ◽

pp. 12816-12826 ◽

Cited By ~ 191

Author(s):

Soila Sukupolvi-Petty ◽

S. Kyle Austin ◽

Whitney E. Purtha ◽

Theodore Oliphant ◽

Grant E. Nybakken ◽

...

Keyword(s):

Dengue Virus ◽

Surface Display ◽

Viral Envelope ◽

Yeast Surface Display ◽

Neutralizing Activity ◽

Denv Serotypes ◽

E Protein ◽

Lateral Ridge ◽

Domain Iii

ABSTRACT Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.

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Genotype-Specific Neutralization and Protection by Antibodies against Dengue Virus Type 3

Journal of Virology ◽

10.1128/jvi.01190-10 ◽

2010 ◽

Vol 84 (20) ◽

pp. 10630-10643 ◽

Cited By ~ 106

Author(s):

James D. Brien ◽

S. Kyle Austin ◽

Soila Sukupolvi-Petty ◽

Katie M. O'Brien ◽

Syd Johnson ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Surface Display ◽

Viral Envelope ◽

Yeast Surface Display ◽

E Protein ◽

Lateral Ridge ◽

Dengue Virus Type 3 ◽

Domain Iii ◽

Positive Strand Rna ◽

Type 3

ABSTRACT Dengue viruses (DENV) comprise a family of related positive-strand RNA viruses that infect up to 100 million people annually. Currently, there is no approved vaccine or therapy to prevent infection or diminish disease severity. Protection against DENV is associated with the development of neutralizing antibodies that recognize the viral envelope (E) protein. Here, with the goal of identifying monoclonal antibodies (MAbs) that can function as postexposure therapy, we generated a panel of 82 new MAbs against DENV-3, including 24 highly neutralizing MAbs. Using yeast surface display, we localized the epitopes of the most strongly neutralizing MAbs to the lateral ridge of domain III (DIII) of the DENV type 3 (DENV-3) E protein. While several MAbs functioned prophylactically to prevent DENV-3-induced lethality in a stringent intracranial-challenge model of mice, only three MAbs exhibited therapeutic activity against a homologous strain when administered 2 days after infection. Remarkably, no MAb in our panel protected prophylactically against challenge by a strain from a heterologous DENV-3 genotype. Consistent with this, no single MAb neutralized efficiently the nine different DENV-3 strains used in this study, likely because of the sequence variation in DIII within and between genotypes. Our studies suggest that strain diversity may limit the efficacy of MAb therapy or tetravalent vaccines against DENV, as neutralization potency generally correlated with a narrowed genotype specificity.

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Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis

PLoS ONE ◽

10.1371/journal.pone.0095263 ◽

2014 ◽

Vol 9 (4) ◽

pp. e95263 ◽

Cited By ~ 21

Author(s):

Aneela Fatima ◽

Haiying Wang ◽

Keren Kang ◽

Liliang Xia ◽

Ying Wang ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Dengue Virus ◽

Virus Type ◽

Immunological Diagnosis ◽

Vhh Antibodies ◽

Dengue Virus Type 2

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Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1519623112 ◽

2015 ◽

Vol 112 (47) ◽

pp. E6506-E6514 ◽

Cited By ~ 142

Author(s):

Roy L. Maute ◽

Sydney R. Gordon ◽

Aaron T. Mayer ◽

Melissa N. McCracken ◽

Arutselvan Natarajan ◽

...

Keyword(s):

Cell Death ◽

Monoclonal Antibodies ◽

Programmed Cell Death ◽

Pet Imaging ◽

Clinical Success ◽

Surface Display ◽

Yeast Surface Display ◽

High Affinity ◽

Signaling Axis ◽

Immune Diagnostics

Signaling through the immune checkpoint programmed cell death protein-1 (PD-1) enables tumor progression by dampening antitumor immune responses. Therapeutic blockade of the signaling axis between PD-1 and its ligand programmed cell death ligand-1 (PD-L1) with monoclonal antibodies has shown remarkable clinical success in the treatment of cancer. However, antibodies have inherent limitations that can curtail their efficacy in this setting, including poor tissue/tumor penetrance and detrimental Fc-effector functions that deplete immune cells. To determine if PD-1:PD-L1–directed immunotherapy could be improved with smaller, nonantibody therapeutics, we used directed evolution by yeast-surface display to engineer the PD-1 ectodomain as a high-affinity (110 pM) competitive antagonist of PD-L1. In contrast to anti–PD-L1 monoclonal antibodies, high-affinity PD-1 demonstrated superior tumor penetration without inducing depletion of peripheral effector T cells. Consistent with these advantages, in syngeneic CT26 tumor models, high-affinity PD-1 was effective in treating both small (50 mm3) and large tumors (150 mm3), whereas the activity of anti–PD-L1 antibodies was completely abrogated against large tumors. Furthermore, we found that high-affinity PD-1 could be radiolabeled and applied as a PET imaging tracer to efficiently distinguish between PD-L1–positive and PD-L1–negative tumors in living mice, providing an alternative to invasive biopsy and histological analysis. These results thus highlight the favorable pharmacology of small, nonantibody therapeutics for enhanced cancer immunotherapy and immune diagnostics.

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Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2011.04.139 ◽

2011 ◽

Vol 409 (2) ◽

pp. 253-259 ◽

Cited By ~ 24

Author(s):

Thomas Han ◽

Jianhua Sui ◽

Andrew S. Bennett ◽

Robert C. Liddington ◽

Ruben O. Donis ◽

...

Keyword(s):

Influenza Virus ◽

Monoclonal Antibodies ◽

Epitope Mapping ◽

Surface Display ◽

Yeast Surface Display ◽

H5n1 Influenza Virus ◽

Highly Pathogenic ◽

H5n1 Influenza ◽

Yeast Surface ◽

Pathogenic H5n1

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Dengue virus envelope domain III immunization elicits predominantly cross-reactive, poorly neutralizing antibodies localized to the AB loop: implications for dengue vaccine design

Journal of General Virology ◽

10.1099/vir.0.055178-0 ◽

2013 ◽

Vol 94 (10) ◽

pp. 2191-2201 ◽

Cited By ~ 16

Author(s):

Xiao-Quan Li ◽

Li-Wen Qiu ◽

Yue Chen ◽

Kun Wen ◽

Jian-Piao Cai ◽

...

Keyword(s):

Antibody Response ◽

Dengue Virus ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Surface Display ◽

Yeast Surface Display ◽

Protein Domain ◽

Dengue Vaccine ◽

Denv Serotypes ◽

Domain Iii

Dengue virus (DENV) is a mosquito-borne virus that causes severe health problems. An effective tetravalent dengue vaccine candidate that can provide life-long protection simultaneously against all four DENV serotypes is highly anticipated. A better understanding of the antibody response to DENV envelope protein domain III (EDIII) may offer insights into vaccine development. Here, we identified 25 DENV cross-reactive mAbs from immunization with Pichia pastoris-expressed EDIII of a single or all four serotype(s) using a prime–boost protocol, and through pepscan analysis found that 60 % of them (15/25) specifically recognized the same highly conserved linear epitope aa 309–320 of EDIII. All 15 complex-reactive mAbs exhibited significant cross-reactivity with recombinant EDIII from all DENV serotypes and also with C6/36 cells infected with DENV-1, -2, -3 and -4. However, neutralization assays indicated that the majority of these 15 mAbs were either moderately or weakly neutralizing. Through further epitope mapping by yeast surface display, two residues in the AB loop, Q316 and H317, were discovered to be critical. Three-dimensional modelling analysis suggests that this epitope is surface exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially on the surface of the mature virion. It is concluded that EDIII as an immunogen may elicit cross-reactive mAbs toward an epitope that is not exposed on the virion surface, therefore contributing inefficiently to the mAbs neutralization potency. Therefore, the prime–boost strategy of EDIII from a single serotype or four serotypes mainly elicited a poorly neutralizing, cross-reactive antibody response to the conserved AB loop of EDIII.

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Antibody Recognition and Neutralization Determinants on Domains I and II of West Nile Virus Envelope Protein

Journal of Virology ◽

10.1128/jvi.01732-06 ◽

2006 ◽

Vol 80 (24) ◽

pp. 12149-12159 ◽

Cited By ~ 214

Author(s):

Theodore Oliphant ◽

Grant E. Nybakken ◽

Michael Engle ◽

Qing Xu ◽

Christopher A. Nelson ◽

...

Keyword(s):

West Nile Virus ◽

Surface Display ◽

Yeast Surface Display ◽

Virus Envelope ◽

West Nile ◽

Lower Efficiency ◽

E Protein ◽

Lateral Ridge ◽

Fusion Loop ◽

Neutralizing Epitopes

ABSTRACT Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E protein. Moreover, in cells expressing Fc γ receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E protein variants, we identified 25 E protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.

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Dengue virus type 2 infects human endothelial cells through binding of the viral envelope glycoprotein to cell surface polypeptides

Journal of General Virology ◽

10.1099/vir.0.19308-0 ◽

2003 ◽

Vol 84 (11) ◽

pp. 3095-3098 ◽

Cited By ~ 32

Author(s):

Hui-Yong Wei ◽

Li-Fang Jiang ◽

Dan-Yun Fang ◽

Hui-Yu Guo

Keyword(s):

Endothelial Cells ◽

Cell Surface ◽

Dengue Virus ◽

Virus Type ◽

Envelope Glycoprotein ◽

Viral Envelope ◽

Human Endothelial Cells ◽

Viral Envelope Glycoprotein ◽

Dengue Virus Type 2

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A New Quaternary Structure Epitope on Dengue Virus Serotype 2 Is the Target of Durable Type-Specific Neutralizing Antibodies

mBio ◽

10.1128/mbio.01461-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 55

Author(s):

E. N. Gallichotte ◽

D. G. Widman ◽

B. L. Yount ◽

W. M. Wahala ◽

A. Durbin ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Dengue Virus ◽

Neutralizing Antibodies ◽

Quaternary Structure ◽

Natural Infection ◽

Viral Envelope ◽

Protective Response ◽

Denv Serotypes ◽

Virus Serotype ◽

Dengue Virus Serotype 2

ABSTRACT Dengue virus serotype 2 (DENV2) is widespread and responsible for severe epidemics. While primary DENV2 infections stimulate serotype-specific protective responses, a leading vaccine failed to induce a similar protective response. Using human monoclonal antibodies (hMAbs) isolated from dengue cases and structure-guided design of a chimeric DENV, here we describe the major site on the DENV2 envelope (E) protein targeted by neutralizing antibodies. DENV2-specific neutralizing hMAb 2D22 binds to a quaternary structure epitope. We engineered and recovered a recombinant DENV4 that displayed the 2D22 epitope. DENV2 neutralizing antibodies in people exposed to infection or a live vaccine tracked with the 2D22 epitope on the DENV4/2 chimera. The chimera remained sensitive to DENV4 antibodies, indicating that the major neutralizing epitopes on DENV2 and -4 are at different sites. The ability to transplant a complex epitope between DENV serotypes demonstrates a hitherto underappreciated structural flexibility in flaviviruses, which could be harnessed to develop new vaccines and diagnostics. IMPORTANCE Dengue virus causes fever and dengue hemorrhagic fever. Dengue serotype 2 (DENV2) is widespread and frequently responsible for severe epidemics. Natural DENV2 infections stimulate serotype-specific neutralizing antibodies, but a leading DENV vaccine did not induce a similar protective response. While groups have identified epitopes of single monoclonal antibodies (MAbs), the molecular basis of DENV2 neutralization by polyclonal human immune sera is unknown. Using a recombinant DENV displaying serotype 2 epitopes, here we map the main target of DENV2 polyclonal neutralizing antibodies induced by natural infection and a live DENV2 vaccine candidate. Proper display of the epitope required the assembly of viral envelope proteins into higher-order structures present on intact virions. Despite the complexity of the epitope, it was possible to transplant the epitope between DENV serotypes. Our findings have immediate implications for evaluating dengue vaccines in the pipeline as well as designing next-generation vaccines.

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