Therapeutic efficacy of humanized monoclonal antibodies targeting dengue virus nonstructural protein 1 in the mouse model

Dengue virus (DENV) which infects about 390 million people per year in tropical and subtropical areas manifests various disease symptoms, ranging from fever to life-threatening hemorrhage and even shock. To date, there is still no effective treatment for DENV disease, but only supportive care. DENV nonstructural protein 1 (NS1) has been shown to play a key role in disease pathogenesis. Recent studies have shown that anti-DENV NS1 antibody can provide disease protection by blocking the DENV-induced disruption of endothelial integrity. We previously demonstrated that anti-NS1 monoclonal antibody (mAb) protected mice from all four serotypes of DENV challenge. Here, we generated humanized anti-NS1 mAbs and transferred them to mice after DENV infection. The results showed that DENV-induced prolonged bleeding time and skin hemorrhage were reduced, even several days after DENV challenge. Mechanistic studies showed the ability of humanized anti-NS1 mAbs to inhibit NS1-induced vascular hyperpermeability and to elicit Fcγ-dependent complement-mediated cytolysis as well as antibody-dependent cellular cytotoxicity of cells infected with four serotypes of DENV. These results highlight humanized anti-NS1 mAb as a potential therapeutic agent in DENV infection.     

Pediatric measles vaccine expressing a dengue antigen induces durable serotype-specific neutralizing antibodies to dengue virus

Dengue disease is an increasing global health problem that threatens one-third of the world's population. Despite decades of efforts, no licensed vaccine against dengue is available. With the aim to develop an affordable vaccine that could be used in young populations living in tropical areas, we evaluated a new strategy based on the expression of a minimal dengue antigen by a vector derived from pediatric live-attenuated Schwarz measles vaccine (MV). As a proof-of-concept, we inserted into the MV vector a sequence encoding a minimal combined dengue antigen composed of the envelope domain III (EDIII) fused to the ectodomain of the membrane protein (ectoM) from DV serotype-1. Immunization of mice susceptible to MV resulted in a long-term production of DV1 serotype-specific neutralizing antibodies. The presence of ectoM was critical to the immunogenicity of inserted EDIII. The adjuvant capacity of ectoM correlated with its ability to promote the maturation of dendritic cells and the secretion of proinflammatory and antiviral cytokines and chemokines involved in adaptive immunity. The protective efficacy of this vaccine should be studied in non-human primates. A combined measles-dengue vaccine might provide a one-shot approach to immunize children against both diseases where they co-exist.     

Protection of mice against Japanese encephalitis virus by passive administration with monoclonal antibodies

We have identified and characterized nine antigenic epitopes on the E envelope of Japanese encephalitis virus (JEV) by using mAb. Passive administration of most of the anti-JEV mAb protected mice from i.v. challenge with 1.5 x 10(3) plaque-forming units of JEV, JaGAr-01 strain. Some mAb, which possess high neutralization activity in vitro, showed high protection, and JEV-specific N mAb 503 was found the most protective. Even an injection of 2.5 micrograms/mouse of mAb 503 protected all mice from JEV infection. Furthermore, an injection of about 200 micrograms of mAb 503 on day 5 postinfection protected 82% of the mice, even when JEV was detected in more than 85% of the infected mouse brains. Synergism of protection was observed with mixtures of several mAb directed against different epitopes. Although in a murine macrophage cell line, all of the mAb groups showed antibody-dependent enhancement (ADE) of JEV infectivity in vitro, and only two flavivirus cross-reactive mAb groups showed ADE of dengue virus type 2. The ADE of JEV by mAb seems not to be harmful for in vivo protection experiments, except for two mAb groups: mAb 302 and 201 showed little or no protective activity against JEV infection and, rather, caused early death in infected mice.     

Mechanism of dengue virus broad cross-neutralization by a monoclonal antibody

The dengue virus (DENV) complex is composed of four distinct but serologically related flaviviruses, which together cause the present-day most important emerging viral disease. Although DENV infection induces lifelong immunity against viruses of the same serotype, the antibodies raised appear to contribute to severe disease in cases of heterotypic infections. Understanding the mechanisms of DENV neutralization by antibodies is, therefore, crucial for the design of vaccines that simultaneously protect against all four viruses. Here, we report a comparative, high-resolution crystallographic analysis of an "A-strand" murine monoclonal antibody, Mab 4E11, in complex with its target domain of the envelope protein from the four DENVs. Mab 4E11 is capable of neutralizing all four serotypes, and our study reveals the determinants of this cross-reactivity. The structures also highlight the mechanism by which A-strand Mabs disrupt the architecture of the mature virion, inducing premature fusion loop exposure and concomitant particle inactivation.     

Temperature-dependent binding of monoclonal antibodies to C hordein

The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5°C but not at 37°C, as determined by enzyme-linked immunosorbent assay (ELISA). The K_d of IFRN 0614 for the consensus peptide was found to be 1.2×10¹² mol⁻¹ at 12°C, but no constant could be calculated at 37°C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and β-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12°C.     

Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody.

Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host. In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5. First, an Escherichia coli expression vector containing a heat-inducible lambda pL promoter was used to synthesize several truncated, and near-full length E polypeptides. Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422. For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR). The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR. The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397). A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay. In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.     

Wild dengue virus types 1, 2 and 3 viremia in rhesus monkeys

Among the flaviviruses, dengue, with its four serotypes, has spread throughout the tropics. The most advanced vaccines developed so far include live attenuated viruses, which have been tested in humans but none has been licensed. Preclinical testing of dengue vaccine candidates is performed initially in mice and in nonhuman primates. In the latter the main criteria used to assay protection are neutralizing antibodies elicited by the vaccine candidate and the magnitude and duration of peripheral viremia upon challenge of previously immunized animals. Towards the identification of wild-type viruses that could be used in challenge experiments a total of 31 rhesus monkeys were inoculated subcutaneously of wild dengue types 1, 2, and 3 viruses. The viremia caused by the different viruses was variable but it was possible to identify dengue viruses useful as challenge strains.     

Human monoclonal antibodies to neutralize all dengue virus serotypes using lymphocytes from patients at acute phase of the secondary infection

The global spread of the four dengue virus serotypes (DENV-1 to -4) has made this virus a major and growing public health concern. Generally, pre-existing neutralizing antibodies derived from primary infection play a significant role in protecting against subsequent infection with the same serotype. By contrast, these pre-existing antibodies are believed to mediate a non-protective response to subsequent heterotypic DENV infections, leading to the onset of dengue illness. In this study, we prepared hybridomas producing human monoclonal antibodies (HuMAbs) against DENV using peripheral blood mononuclear cells (PBMCs) from patients in the acute phase (around 1 week after the onset of illness) or the convalescent phase (around 2weeks after the onset of illness) of secondary infection. Interestingly, a larger number of hybridoma clones was obtained from patients in the acute phase than from those in the convalescent phase. Most HuMAbs from acute-phase infections were cross-reactive with all four DENV serotypes and showed significant neutralization activity to all four DENV serotypes. Thus, secondary DENV infection plays a significant role in stimulating memory cells to transiently increase the number of antibody-secreting plasma cells in patients in the early phase after the secondary infection. These HuMAbs will enable us to better understand the protective and pathogenic effects of DENV infection, which could vary greatly among secondarily-infected individuals.     

Hybridomas producing monoclonal antibodies to Crimean hemorrhagic fever virus

A series of hybridomas producing monoclonal antibodies to Crimean hemorrhagic fever virus was obtained and their cultural properties were characterized. HEMA-12 and HEMA-24 secreted monoclonal IgG2b antibodies, HEMA-101 secreted monoclonal IgG1 antibodies, HEMA-31, HEMA-9 and HEMA-11 secreted monoclonal IgG2 antibodies. According to the results of the indirect immunofluorescence test, the titer of specific immunoglobulins in the culture fluid was 1:16-1:32, but sometimes reached 1:64-1:128. The titer of antibodies in ascitic fluid amounted to dilutions of 10(4)-10(5). Hybridomas were cloned by the method of ultimate dilutions. The injection of 5-15 million HEMA cells into the abdominal cavity of BALB/c mice induced the formation of ascitic tumors in the animals within 7-11 days and the accumulation of ascitic fluid in a volume of 1-4 ml. Hybridomas, found to be capable of passage from mouse to mouse, underwent 5-16 passages during the term of observation.     

Mapping the binding of monoclonal antibodies to histone H5

The binding sites of nine monoclonal antibodies along the polypeptide chain of histone H5 were mapped. Immunoblotting experiments with peptides generated from H5 by trypsin digestion, N-bromosuccinimide cleavage, and cyanogen bromide cleavage revealed that all of the monoclonal antibodies reacted with the globular region of H5 which is encompassed by amino acid residues 22-98. Within this globular segment, the epitopes could be subdivided into three regions. Monoclonals 1G11, 2E5, and 2H5 bind to residues 28-31. The close proximity of the epitopes was verified by a competitive enzyme-linked immunosorbent assay and by their binding pattern to a tryptic digest of H5. Monoclonals 4C6, 6E12, and 2E12 bind to a region encompassed by amino acids 28-53 while monoclonals 4H7, 1C3, and 3H9 bind to a region encompassed by residues 53-98. Precise localization of the epitopes in the primary sequence of H5 will allow detailed studies on the mode of binding of H5 to core particles in chromatin.     

Analysis of epitopes on dengue virus envelope protein recognized by monoclonal antibodies and polyclonal human sera by a high throughput assay

Background

The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored.

Methodology/Principal Findings

We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer.

Conclusions/Significance

Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.     

Nematospiroides dubius: passive transfer of protective immunity to mice with monoclonal antibodies

Nine hybridoma cell lines secreting monoclonal antibodies specific for Nematospiroides dubius were produced by fusion of the mouse myeloma cell line NS-1 to either spleen cells or mesenteric lymph node cells from mice repeatedly infected with N. dubius. Seven of the antibodies were identified as IgM and two as IgG1. Each monoclonal antibody bound to polypeptide epitopes on both infective larvae (L3) and adult worms. However, five antibodies bound preferentially to L3 and three to adult worms. All nine antibodies reacted with high molecular weight protein antigens. Passive protective immunity in Balb/c mice was demonstrated with monoclonal antibodies Nd2 and Nd3 in ascites fluid which stunted both male and female worms and reduced parasite fecundity.     

Anti-idiotypic monoclonal antibodies to GM1 identify ganglioside binding proteins

Ganglioside stimulated neurite outgrowth may be due to gangliosidebinding to membrane proteins or to intercalation into the membrane.To test that ganglioside binding proteins could be found onneuronal surfaces, antiidiotypic ganglioside monoclonal antibodies(AIG mAbs) were generated to mimic the biological propertiesof the GM1 ganglioside. The AIG mAbs were identified by theirability to bind to a known GM1 binding protein, the ß-subunit of cholera toxin. For the two AIG mAbs studied, AIG5 andAIG20, binding to ß-CT was blocked most strongly byGM1. This data also suggests that AIG5 and AIG20 mimic differentbut overlapping epitopes of the ganglioside GM1. Western blottingand immunoprecipitation of mammalian tissues reveals four potentialganglioside binding proteins of molecular weight 93, 66, 57,and 45 kDa. Immunocytochemistry demonstrates neuronal surfacelabel with the AIG mAbs, which suggests that gangliosides, enrichedon the neuronal surface membrane, are co-localized with putativeganglioside binding proteins. In bioassays, the AIG mAbs promoteneuronal sprouting. This shows that these antibodies can beused to study the biological effects of ganglioside bindingto neuronal surface proteins, and the role of gangliosides inthe activation of neurite outgrowth. agonist antibody anti-idiotypic antibody gangliosides ganglioside binding proteins     

百合无症病毒单克隆抗体的制备及检测应用

用百合无症病毒(Lilysymptomlessvirus,LSV)免疫的BALBC鼠脾细胞与SP20鼠骨髓瘤细胞融合,经筛选克隆,获得4株能稳定传代并分泌抗LSV单克隆抗体(MAb)的杂交瘤细胞(2A2、5H9、5H2和5E12),并分别制备它们的单抗腹水。4株单克隆抗体腹水间接ELISA效价达10-6,5H9和5E12的抗体类型及亚类均为IgG1,而2A2和5H2均为IgG3,4株单克隆抗体的轻链均为κ链。利用单克隆抗体建立了抗原包被间接ELISA(ACP-ELISA)检测LSV的方法。病叶作1300倍稀释、提纯LSV病毒浓度为18ngmL(每孔的病毒绝对量为1.8ng)时,该方法仍能检测到病毒。利用ACP-ELISA检测了田间样品,发现LSV在百合上发病很普遍。     

Production of monoclonal antibodies to Tomato leaf curl Bangalore virus

Purified Tomato leaf curl Bangalore virus (ToLCBV) was injected into mice and the splenocytes were used for establishing hybridoma lines. Initial screening of culture supernatants showed that 13 lines produced antibody, and after further screening four produced functional monoclonal antibodies. Upon characterisation, these were found to be of low affinity, probably due to host protein contamination and poor yield of native virus in the original preparations. In order to circumvent these problems, the coat protein of ToLCBV was over-expressed in Escherichia coli. Fusion experiments using recombinant coat protein as antigen yielded two primary hybridoma clones G₁₁ and E4 that exhibited good affinity of binding to the antigen. Sub-cloning yielded four monoclonal antibodies G₁₁E₇E₇, G₁₁E₇G₁₂, E₄E₂ and E₄G₆. G₁₁E₇E₇ and G₁₁E₇G₁₂ successfully detected ToLCBV in infected leaf extracts of tomato and Nicotiana benthamiana, viruliferous whiteflies and weed samples. These monoclonal antibodies could also detect other type III geminiviruses such as Pumpkin yellow vein mosaic virus and Bhendi yellow vein mosaic virus. Thus these monoclonal antibodies can be used for testing field-collected samples.     

Biological characterization of human monoclonal antibodies to rabies virus.

Rabies virus antigen-specific human monoclonal antibodies (MAbs) that recognized either viral glycoprotein, ribonucleoprotein, or matrix proteins were generated. Only glycoprotein-specific MAb neutralized a variety of rabies viruses and protected laboratory rodents against lethal rabies virus infection. The determinant recognized by this MAb does not appear to reside in previously defined antigenic sites of the viral glycoprotein.     

Development and characterization of monoclonal antibodies to Ebola virus glycoprotein

Balb/С mice were immunized with recombinant Ebola virus glycoprotein. Following the selection, screening, and cloning of murine hybridomas, we obtained five genetically stable clones of monoclonal antibodies GPE118 (IgG), GPE274 (IgM), GPE325 (IgM), GPE463 (IgM), and GPE534 (IgG). These antibodies were isolated and purified from the ascitic fluid of Balb/С mice using Protein G affinity chromatography (for IgG) and euglobulin precipitation (for IgM). To select at least three candidate antibodies for testing in biological assays as components of an antibody cocktail for the prophylaxis and treatment of hemorrhagic fever, we carried out an immunochemical analysis of the epitope specificity of the isolated antibodies. Based on the data of immunoblotting and sandwich ELISA, it became evident that the epitope recognized by GPE 534 differs from the epitopes recognized by the monoclonal antibodies GPE 118 and GPE 325. The last two antibodies also have different epitope specificity: it follows from the immunoblotting data and from the data on the binding of these antibodies with the intact and oxidized (partly deglycosylated) recombinant glycoprotein. For the biological activity studies and the development of recombinant counterparts, we selected three candidate high-affinity monoclonal antibodies GPE 534, GPE 118, and GPE 325.