| Dengue virus (DENV) is a mosquito-borne virus that causes human dengue diseases, ranging from asymptomatic infections, dengue fever (DF) to more severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). The burden of dengue diseases is on the rise in tropical and subtropical areas, especially in South America, South-east Asia and Caribbean regions. There are also more and more reports of dengue diseases in Africa and the Middle East. In Southern China, the epidemics of DF occur almost each year. The rigorous situations of dengue epidemic arouse much concern, however, its pathogenesis is far from elucidation. It is accepted that virus itself, immunity history of the host and individual genetic background all play important roles in the progress of the severe dengue diseases. Among these factors, the role that dengue virus itself plays is critical. The primary infection caused by any of the four serotypes of DENV probably predisposes individuals to display increased viral loads, shorten latent period and accordingly elevated risk of progressing into DHF/DSS when the individual is infected by a serotype different from that causing primary infection. The severity of diseases are associated with the serotype, genotype, virus sequences of DENV causing secondary infection and as well as the interval between the primary and secondary infection.To deal with the ever-increasing dengue diseases, many research organizations speed up their efforts in developing effective dengue vaccine candidates. At present, there are already several kinds of vaccines that are at preclinical levels, including live-attenuated vaccines, inactivated virus vaccines, subunit vaccines based on recombinant proteins and naked DNA constructs. Since there is no licensed vaccine available on the basis of traditional live attenuated vaccines, researchers focused more on immune strategy of recombinant subunit vaccines. Though developing dengue vaccine candidates is feasible, considerable challenges present:(1) Dengue vaccines must provide protection against infections caused by all four serotypes of DENV simultaneously, thereby making necessary development of tetravalent vaccines;(2) Dengue vaccines can provide long-term protection, because there is report that DHF developed when re-infection occurred20years after the primary infection;(3) There is still no suitable animal models to replicate dengue diseases;(4) Though the in vitro protective role of neutralizing antibodies is widely accepted, their relationship with practical protection still needs determination;(5) At the time of changes of transmission patterns and viral strains of DENV, dengue vaccines need re-evaluation. Thus, to develop reliable dengue vaccines, it is essential to address two issues:one is the efficacy of vaccines, namely the protection of vaccines, and the other is safety issue of vaccines.The envelope of DENV consists of lipid bilayer, containing two kinds of envelope-associated proteins:envelop protein (E) and membrane protein (M). E protein involves viral attachment, and fusion with endosome after entry via binding to cellular receptors. E protein contains three different domains, domain Ⅰ (EDⅠ), EDⅡ and EDⅢ. EDⅢ is surface exposed on the virion and accessible, and recombinant EDIII can inhibit the infectivity of the virus. A line of evidences demonstrated that EDIII contains type-specific (TS) epitopes against a single DENV serotype, sub-complex reactive (sCR) epitopes against two or three DENV serotypes, and complex reactive epitopes (CR) against all four serotypes. A number of murine monoclonal antibodies (mAbs) localize to these epitopes. Many studies show that some TS and sCR murine mAbs are mainly neutralizing and protective mAbs against DENV, accordingly, EDIII protein of DENV has become a promising candidate target of subunit vaccines. In addition, mAbs against EDIII are shown recently to take up a small part in the total DENV-specific antibodies in human immune sera, and the epitopes on EDIII are not the main targets of human neutralizing antibodies against DENV. In spite of this, the antigenic sites of murine mAbs and their neutralizing activity spectrum are far from making clear. Further studies are essential to gain deeper insight into the complexity of humoral immune reactions, the optimization of immune strategy of dengue vaccines and the study of therapeutic antibody.The studies of antigenic epitopes and neutralizing potency of mAbs directed against EDIII of DENV are instrumental to efficacy of dengue vaccines, but safety issue of dengue vaccines is another difficulty. As mentioned above, after the primary infection by any of the four serotypes, the risk of development into severe dengue diseases is significantly increased in the case of secondary infection caused by a heterotypic serotype. One of the main underlying mechanisms is antibody-dependent enhancement (ADE). Pre-existing cross-reactive weakly neutralizing antibodies bind to virus and enhance the infectious level of cells bearing Fc gamma receptor (FcyR). Recently, the study of Dejnirattisai W. and colleagues present the complexity of ADE because both anti-E and anti-prM (precursor membrane) antibodies can enhance DENV infection. Since it is urgent to develop tetravalent vaccines that provide long-term protection and there is no appropriate animal models for the evaluation of safety issues of dengue vaccines, the evaluation of dengue vaccines in vitro appears critically important.One hundred and seven strains of mAbs were produced using recombinant EDⅢ protein as immunogens to immune BALB/c mice in previous studies of our laboratory. In the current study, we first identified the binding specificity or cross-reactivity of107mAbs utilizing enzyme-linked immuno-sorbent assay (ELISA) and immunofluorescence assay (IFA), followed by the measurement of the neutralizing activities of94strains mAbs selected using established enzyme-linked immuno-spot mini-neutralizing test (ELISPOT-MNT). On the basis of these, we identified and analyzed the antigenic sites of94mAbs to know further the antigenic sites recognized by mAbs obtained by immune of recombinant EDⅢ and their relationship with protective roles of these mAbs in vitro. Then, we develop a simple and high-throughput alternative assay for ADE determination to assess the safety issue of dengue vaccines and the risk of dengue diseases.Therefore, the three main aims of this study are as follows:First, to identify the binding specificity or cross-reactivity and neutralizing activities of107mAbs. Second, to analyze the antigenic sites recognized by tested mAbs in this study. Third, to develop a simple and high-throughput alternative assay for ADE measurement to realize the evaluation of the safety issue of dengue vaccines in vitro.This study is composed of three parts:Part Ⅰ Characterization of binding capacity/and neutralizing activities of monoclonal antibodies against E protein domain Ⅲ of Dengue virusIn previous studies of our laboratory, we produced107strains of mAbs by expressing recombinant EDⅢ of each DENV serotype by Pichia pastoris, and immunizing BALB/c mice intraperitoneally with each of the recombinant EDⅢ proteins alone, or a combination of recombinant EDⅢs of all four serotypes. In this study, characterized in detail the binding specificity or cross-reactivity with and neutralizing activities of these mAbs against different serotypes of DENVs. ELISA and IFA showed that94strains of mAbs exhibited binding specificity or cross-reactivity with recombinant EDⅢ proteins and DENV-infected C6/36cells, including42strains of TS mAbs,26strains of sCRs mAbs,23strains of CR mAbs and3strains of flavivirus cross-reactive mAbs. Of note, the binding specificity or cross-reactivity identified by IFA was not completely consistent with that identified by ELISA, probably due to the conformational discrepancy between coated recombinant EDⅢ protein and surface-exposed E protein on the infecting DENV virions.Then, with the enzyme-linked immunospot based mini-neutralization test (ELISPOT-MNT) established previously, we determined the neutralizing activities in vitro of these mAbs against all four DENV serotypes. According to the50%inhibition concentration (IC50), mAbs were designated strong (IC50≤1μg/ml), moderate (IC50between1and50μg/ml) and weak (IC50>50μg/ml) neutralizing activities. Seventeen DENV-1TS mAbs and14DENV-2TS mAbs showed strongly, moderately or weakly neutralizing activity towards a single serotype. All of the7DENV-2TS mAbs displayed no neutralizing activity against DENV-2. All of the3DENV-3TS mAbs exhibited strong neutralizing activity against DENV-3. The panel of26sCR mAbs and23CR mAbs displayed a varied repertoire of neutralizing activity towards the four DENV serotypes, with strongly, moderately or weakly neutralizing activity towards different serotypes. It was found that the binding specificity or cross-reactivity and neutralizing potency was not always consistent. Such inconsistence may due to the conformational difference between isolated recombinant EDIII and E protein displayed on the surface of natural viral particles. Our results identified clearly the binding specificity or cross-reactivity and in vitro neutralizing capacity of our panel of mAbs, laying a good foundation for further study of antigenic sites of these mAbs, deeper structural studies and full functional determination.Part II Characterization of epitopes in envelope protein domain III of dengue virus recognized by monoclonal antibodiesE protein domain Ⅲ (EDⅢ) is considered a promising region for subunit vaccine candidates. However, the antigenic sites in the EDⅢ and their relationship with protective roles in vitro of mAbs elicited using recombinant EDⅢ as immunogens have not been clearly defined.In this study, based on the binding specificity or cross-reactivity with and neutralizing activities of94mAbs against different serotypes of DENVs, we characterized in detail the antigenic sites recognized by these mAbs. First, through pepscan using synthetic overlapping peptides, we found that12mAbs consisting of2DENV-2type-specific mAbs,5sub-complex reactive mAbs, one complex reactive mAb and2flavivirus cross-reactive mAbs specifically recognized different linear epitopes and display no neutralizing activity against any serotype. In contrast, we observed that two third (15of23) complex-reactive mAbs predominantly recognized the same amino acid sequence309-320. Further sequence alignment amino acid substitution analysis demonstrated that aa309-320is a DENV complex reactive epitope that is immunodominant and highly conserved epitope across different serotypes of dengue virus.Of note, all15complex-reactive mAbs exhibited significant cross-reactivity with coated EDIII from all DENV serotypes in ELISA and also with DENV-1,-2,-3, and-4infected C6/36cells in IFA. However, neutralization tests indicated that the majority of these15mAbs were either moderate or weak neutralizers. How to account for such difference between significant cross-reactivity and neutralizing capacity exhibited by these15mAbs? Through further identification of this epitope by site-directed mutagenesis and yeast surface display mapping, two residues on the AB loop, Q316and H317, were discovered to be critical. Three-dimensional modeling analysis of the310-319epitope in the crystal structures of the DENV E protein suggests that this antigenic site is surface-exposed on EDIII but less accessible on the surface of the E protein dimer and trimer, especially in tightly packed E protein on the surface of the mature virion. It is concluded that EDIII as immunogen may elicit mAbs to the epitope that is not exposed on the virion surface, therefore contributing inefficiently to their neutralization potency. With this study, we reasonably provide explanation for the relationship between poorly neutralizing cross-reactive antibody response to AB loop of EDIII and EDIII prime-boost strategy. We thus gained deeper insight into the EDIII-based subunit immune strategy and provided data for further exploration of optimized EDIII-based vaccination regimens.Part Ⅲ Development of simple high-throughput assays to evaluate the function of antibodies against dengue virusDevelopment of dengue vaccines entails minimizing potential antibody-dependent enhancement (ADE) and achieving an even protective antibody response against all four dengue virus (DENV) serotypes. In the absence of appropriate animal models, it is necessary to develop reliable and high-throughput platforms to determine the immune status of the disease process and vaccine efficacy,In this study, using FcyR-expressing K562cells and a known enhancing antibody, we creatively developed a simple high-throughput ADE assay of enhancing activity of DENV-specific antibodies by quantitative measurement of NS1antigen in the culture supernatant using NS1capture ELISA. To evaluate this method, enhancing activity was measured simultaneously by two methods, the newly developed increased NS1antigen quantitation and traditional enhanced virus titration. The kinetics of NS1production were compatible with the results of the virus titration assay. Linear correlation analysis demonstrated a good agreement (R=0.938, P=0.000) between NS1and infectious viral titer determination. Then, enhancing and neutralizing antibody activities of two flaviviral cross-reactive murine monoclonal antibodies (mAbs)(4G2and2A10G6) and four primary DENV-1infected human sera were evaluated and analysed by ELISA-ADE and an available enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) both in96-well formats and for easy readout. It was shown that characteristic ADE profiles in a dose-dependent manner and the enhancements occurred at sub-neutralizing concentrations, displaying reasonable associations with the neutralizing effect measured via ELISPOT-MNT.The newly developed simple high-throughput ELISA-ADE and ELISPOT-MNT assays are valuable for the functional evaluation of DENV enhancing and neutralizing antibody activities. These assays can significantly facilitate evaluations of the safety and efficacy of DENV vaccine candidates in large-scale studies and promote the elucidation of immune status during vaccination and dengue disease progression.Summarization:1. In this study, we characterized in detail the binding capacity with different serotypes of DENV and found that94mAbs exhibited binding specificity or cross-reactivity with recombinant EDIII protein and/or DENV-infected C6/36cells. Then, with the established ELISPOT-MNT, we determined the neutralizing activities in vitro of these mAbs against all four DENV serotypes. Half of the42DENV-1type-specific mAbs showed strongly neutralizing activity towards a single serotype. One third of26sub-complex reactive mAbs and two third of23complex reactive mAbs displayed a varied repertoire of neutralizing activity towards the four DENV serotypes, with strongly, moderately or weakly neutralizing activity towards different serotypes. Conformational difference between isolated recombinant EDIII and E protein displayed on the surface of natural viral particles reasonably account for the inconsistence between binding and neutralizing capacity. With this study, we obtained strongly neutralizing mAbs of interest (e.g. mAbs3E31and2D73was used for crystal structure and functional research by other investigator in our laboratory). Our results laid a good foundation for further study of antigenic sites of these mAbs, deeper structural and functional determination, and therapeutic mAbs.2. In this study, on the basis of the study above, we characterized in detail the antigenic sites recognized by these mAbs. Through pepscan, sequence alignment and amino acid substitution, we found that two third (15of23) complex-reactive mAbs predominantly recognized the same amino acid sequence309-320. This is the first report of immunodominance of highly conserved DENV complex reactive epitope aa310-319. Further identification of this epitope by site-directed mutagenesis and yeast surface display mapping, two residues on the AB loop, Q316and H317, were discovered to be critical. With three-dimensional modeling analysis, we concluded that EDIII as immunogen may elicit mAbs to epitope that is not exposed on the virion surface, therefore contributing inefficiently to their neutralization potency. With this study, we reasonably provide explanation for the relationship between poorly neutralizing cross-reactive antibody response to AB loop of EDIII and EDIII prime-boost strategy. We thus gained deeper insight into the EDIII-based subunit immune strategy and provided data for further exploration of optimized EDIII-based vaccination regimens.3. In this study, we developed a simple high-throughput ADE assay (ELISA-ADE) of enhancing activity of DENV-specific antibodies by quantitative measurement of NS1antigen in the culture supernatant using NS1capture ELISA using FcyR-expressing K562cells and a known enhancing antibody. This new assay allows for simple, rapid and high-throughput detection of enhancement activities, while avoiding cumbersome, time-consuming procedures and complex equipment. We present for the first time the newly developed simple high-throughput ELISA-ADE and ELISPOT-MNT assays for the functional evaluation of DENV enhancing and neutralizing antibody activities of mAbs and polyclonal immune sera. These assays can significantly facilitate evaluations of the safety and efficacy of DENV vaccine candidates in large-scale studies and promote the elucidation of immune status during vaccination and dengue disease progression. |
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