Phage Display Approaches for the Isolation of Monoclonal Antibodies Against Dengue Virus Envelope Domain III from Human and Mouse Derived Libraries

Domain III of the dengue virus envelope protein (EDIII, aa295-395) has an immunoglobulin fold and is the proposed receptor-binding domain of the virus. Previous studies have shown that monoclonal antibodies against EDIII can be neutralizing and have therapeutic potential. Here, cloned Fab-phage libraries of human and mouse origin were screened for DENV specific antibodies. Firstly, bacterially expressed EDIII or whole virus particles were used as bait in biopanning against a large naïve human Fab-phage library (>10 billion independent clones). Multiple panning strategies were employed, and in excess of 1000 clones were screened, but all of the antibodies identified bound the envelope in regions outside EDIII suggesting EDIII antibodies are virtually absent from the naïve human repertoire. Next, a chimeric Fab-phage library was constructed from a panel of EDIII specific mouse hybridomas by pooling the VH and VL chain sequences from the hybridomas and cloning these into the pComb3X phagemid vector with human CH and CL encoding sequences. Biopanning against EDIII identified a unique antibody (C9) that cross-reacts with EDIII from DENV1-3 and, in the IgG format, binds and neutralizes DENV2 in cell-based assays. Sequence analysis and saturation mutagenesis of complementary determining regions (CDR) in the C9 light chain suggest an antigen recognition model in which the LCDR3 is a key determinant of EDIII specificity, while modifications in LCDR1 and LCDR2 affect DENV serotype cross-reactivity. Overall, this study supports the current prevailing opinion that neutralizing anti-EDIII monoclonal antibodies can be readily generated in murine systems, but in humans the anti-DENV immune response is directed away from domain III

Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII

Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2

Dengue virus (DENV) infection remains a serious health threat despite the availability of supportive care in modern medicine. Monoclonal antibodies (mAbs) of DENV would be powerful research tools for antiviral development, diagnosis and pathological investigations. Here we described generation and characterization of seventeen mAbs with high reactivity for E protein of DENV. Four of these mAbs showed high neutralizing activity against DENV-2 infection in mice. The monoclonal antibody mAb DB32-6 showed the strongest neutralizing activity against diverse DENV-2 and protected DENV-2-infected mice against mortality in therapeutic models. We identified neutralizing epitopes of DENV located at residues K310 and E311 of viral envelope protein domain III (E-DIII) through the combination of biological and molecular strategies. Comparing the strong neutralizing activity of mAbs targeting A-strand with mAbs targeting lateral ridge, we found that epitopes located in A-strand induced stronger neutralizing activity than those located on the lateral ridge. DB32-6 humanized version was successfully developed. Humanized DB32-6 variant retained neutralizing activity and prevented DENV infection. Understanding the epitope-based antibody-mediated neutralization is crucial to controlling dengue infection. Additionally, this study also introduces a novel humanized mAb as a candidate for therapy of dengue patients

Sulfated Polysaccharide, Curdlan Sulfate, Efficiently Prevents Entry/Fusion and Restricts Antibody-Dependent Enhancement of Dengue Virus Infection In Vitro: A Possible Candidate for Clinical Application

10.1371/journal.pntd.0002188PLoS Neglected Tropical Diseases74

The Development of Therapeutic Antibodies That Neutralize Homologous and Heterologous Genotypes of Dengue Virus Type 1

Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential

Vaccines against Major Poultry Viral Diseases: Strategies to Improve the Breadth and Protective Efficacy

The poultry industry is the largest source of meat and eggs for human consumption worldwide. However, viral outbreaks in farmed stock are a common occurrence and a major source of concern for the industry. Mortality and morbidity resulting from an outbreak can cause significant economic losses with subsequent detrimental impacts on the global food supply chain. Mass vaccination is one of the main strategies for controlling and preventing viral infection in poultry. The development of broadly protective vaccines against avian viral diseases will alleviate selection pressure on field virus strains and simplify vaccination regimens for commercial farms with overall savings in husbandry costs. With the increasing number of emerging and re-emerging viral infectious diseases in the poultry industry, there is an urgent need to understand the strategies for broadening the protective efficacy of the vaccines against distinct viral strains. The current review provides an overview of viral vaccines and vaccination regimens available for common avian viral infections, and strategies for developing safer and more efficacious viral vaccines for poultry

Studies on the inhibition of viral entry of flaviviruses

166 p.Dengue virus serotypes 1-4 (DENV1-4) cause serious febrile illness in humans, including Dengue haemorrhagic fever (DHF) and Dengue shock syndrome (DSS). Presence of avid neutralizing antibodies during clinical illness is critical in the outcome of clinical disease and severity of manifestations. Presently there are no specific anti-flaviviral therapeutic agents or an effective vaccine available for clinical use. In the current study, the possibilities of using the DENV envelope protein domain III (Ed3), as it possesses receptor binding activity, as a possible competitor to whole virus for entry into host cell receptor was explored.DOCTOR OF PHILOSOPHY (SBS

Vaccines against Major Poultry Viral Diseases: Strategies to Improve the Breadth and Protective Efficacy

The poultry industry is the largest source of meat and eggs for human consumption worldwide. However, viral outbreaks in farmed stock are a common occurrence and a major source of concern for the industry. Mortality and morbidity resulting from an outbreak can cause significant economic losses with subsequent detrimental impacts on the global food supply chain. Mass vaccination is one of the main strategies for controlling and preventing viral infection in poultry. The development of broadly protective vaccines against avian viral diseases will alleviate selection pressure on field virus strains and simplify vaccination regimens for commercial farms with overall savings in husbandry costs. With the increasing number of emerging and re-emerging viral infectious diseases in the poultry industry, there is an urgent need to understand the strategies for broadening the protective efficacy of the vaccines against distinct viral strains. The current review provides an overview of viral vaccines and vaccination regimens available for common avian viral infections, and strategies for developing safer and more efficacious viral vaccines for poultry

Inhibition of West Nile virus entry by using a recombinant domain III from the envelope glycoprotein

JOURNAL OF GENERAL VIROLOGY862405-412JGVIAUNITED KINGDOMComplete

Antibodies targeting the PfRH1 binding domain inhibit invasion of Plasmodium falciparum merozoites

10.1371/journal.ppat.1000104PLoS Pathogens47e100010