Neutralizing And Enhancing Activities Of Antibodies Against Envelope Protein Domain Ⅲ Of Dengue Virus

Dengue fever is one of the rapidly spread of vector-borne diseases in the world, and is transmitted by the bite of a mosquito infected with one of the four dengue virus (DENV)serotypes. More than 40 percent of the world population, about 2.5 billion people at risk from dengue. Each of the four serotypes of DENV (DENV-1, DENV-2, DENV-3, and DENV-4) is capable of causing a spectrum of clinical manifestation,range from mild fever to high fever, with severe headache, rash, muscle and joint pain. Severe dengue, which was termed as dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), is characterized by fever, abdominal pain, persistent vomiting, bleeding and breathing difficulty, and is a potentially lethal complication, affecting mainly children. The spread of dengue often occurs in urban and semi-urban areas of tropical and subtropical climates, and it is becoming a major international public health concern. In recent years, dengue epidemic is worsening in Southeast Asia and the Americas.A varying degree of dengue prevalence almost occurred every year in Guangdong, Guangxi, Hainan and Fujian in China, and the majority of cases concentrated in the Guangdong province. In 2014, all four serotypes of the dengue virus had been prevalent in China, but only DENV-1 is most common.Outbreak of dengue fever in Guangzhou in June 2014 was the most severely in the past 20 years. World Health Organization stressedthat there is no licenced vaccine or any specific medicine to treat dengue in 2014. The only prevention way is personal protective measures by reducing and control the spread of mosquitoes, avoiding being bitten.DENV genome is single-stranded RNA strand (approximately 11kb), contains a single open reading frame encoding three structures proteins, namely capsid protein (C), premembrane/membrane protein (prM/M), envelope proteins (E), and seven non-structural protein (NS) namely NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. The surface of the mature DENV particle is covered with 90 head-to-tail homodimers of envelope glycoprotein (E), which is the major surface protein of flaviviruses.The E protein ectodomain can be divided into three distinct structural domains designated as Ⅰ (EDⅠ), Ⅱ (EDⅡ), and Ⅲ (EDⅢ). EDⅢ consists of an immunoglobulin-like fold and is likely involved in receptor binding and neutralizing of antibody.NS1 exists in multiple oligomeric forms and isfound in different cellular locations, showing as cell membrane type, intracellular typeand asecreted extracellular hexamertype. NS1 protein can be considered as an important biomarker for early diagnosis. The discovery of remarkably high levels of circulating NS1 is correlated with severity of DENV infection inpatients. The measurement ofNSl by antigen capture ELISA revealed that NS1 can reach at high levels of upto 50μg/mlin some infected individuals. Therefore, we have taken NS1 level as a parameter to evaluate the severity of infection in present work.The major obstacle for development of vaccine is attributed to that most of vaccine candidates are incapable of provoking the protection for all serotypes DENV infection as well as antibody-dependent enhancement (ADE) can occur in secondary infection with heterotypic DENV.Generally, primary infection with DENV leads to the induction of long-term serum neutralizing antibodies, and this has been reasonably interpreted as persistence of long-term immunity against disease upon reinfection with the same serotype. In prospective studies of populations with endemic dengue, a secondary infection with virus of the same serotype that results in illness is rare. Severe disease (DHF/DSS) predominantly occurs following secondary infection with a heterotypic DENV.Antibody-dependent enhancement (ADE) by pre-existing cross-reactive, non-neutralizing antibodies or subneutralizing antibodies have been considered to play an important role in disease severity by promoting virus entry into FcyR-bearing cells. The presence of ADE impedes the development of dengue vaccines that should induce protective neutralizing antibodies without enhancing viral replication. The ideal DENV vaccinesshouldprotect against each of the four DENV serotypes, provide lifelong protection, and should guarantee thesafety.The amino acid sequence of the E protein in each serotype is well conserved, with a minimum similarity of between 90% and 96%. However, amino acid sequence similarity of the E proteins between different serotypes is generally in the range of 60-70%. The dengue researchesmainly focus on DENV EDⅢ proteins and EDⅢ-reactive antibodies. It isdemonstrated that the most strong neutralizing mouse antibody recognise epitopes in EDⅢ,and the epitope ischaracterized atEDⅢ A strand and lateralridge. Moreover,the epitope recognized by EDⅢ-reactive antibodies in DENV infected human serum and mouse antibodies is similar. Recent studies revealed that EDⅢ-reactive antibodies in convalescent sera from patients infected with dengue are considered to contribute little to neutralizing and enhancing activities. This could be explained that antibodies of patient serum are induced by many different epitopes on complex DENV, and not all of protective epitopes can be as predominant epitope to provoke an effective neutralizing antibody response and even ADE, however, protective non-predominant epitopes presented in purified protein may be as subunite vaccine candidates to induce protective neutralizing antibodies without enhancing viral replication. Our work,as followswould partly demonstrate this presume.Part I. Ascertain of critical residues onDENV EDIII epitopesrecognized by cross-reactive mAbsOur previousstudies prepared a set of anti-DENV-1,-2,-3 and -4 EDⅢ cross-reactive monoclonal antibodies(mAb) using recombinant DENV EDⅢas immunogens. We observed that two third (15 of 23) cross-reactive mAbs predominantly recognized the same amino acid sequence aa310KEVAETQHGT319.However, these mAbs show the strong binding with moderate or weak neutralization. To further clarify the key amino acid residues of the antigen-antibody binding site, DENV EDⅢ protein was expressed on yeast cells using yeast surface display technology, then amino acids mutationof aa310-319 sequences using site-directed mutagenesis techniques was performed. The binding of these mAbs to mutations EDⅢ was detected by flow cytometry. Yeast surface display technology is an effective tool for the study of the interaction between soluble proteins, which can be quickly and easily used to determine the binding and interaction between antigens and antibodies, receptors and ligands. Eukaryotic yeast cell possess the processing of protein modification, and the protein is closer to the native conformation. The site-directed mutagenesis is a routine tool for studying the complex relationship between structure and function. It is simple, fast and efficient translation of DNA sequences to obtain the desired protein or DNA expression to clarify the site of normal and mutant differences. Through further identification,two residues on the AB loop, Q316 and H317, were proved to be critical.It would befoundation for furtheroptimizing and modified EDⅢas subunit vaccine strategy.Part II. Developing simple high-throughput assays to evaluate antibody-dependent enhancement activity on dengue virusThe humoral immune response to dengue viruscan function as protection and the enhancing infection. The primary protective antigen of DENV is the E protein, which also serves as the principal target for neutralizing antibodies. Neutralization could be achieved when enough molecules of antibody bind to the accessible epitopes on the E protein and prevent binding of virus to the target cell and release of virion RNA into the cytoplasm of a susceptible cell. This enhanced virus replication is mediated primarily by pre-existing, non-neutralizing or subneutralizing antibodies to E or prM antigensthat enhanceaccess of virionswith bound antibodies to FcyR-bearing cells.Based oncorrelation between of the levels of circulating NS1 protein and disease severity, our team previously developed an available enzyme-linked immunospot-based micro-neutralization test (ELISPOT-MNT) in 96-well formats and for easy readout.In present work, we established a simple method to evaluate ADE activity by quantitative measurement of NS1 antigen in the culture supernatant by NS1 capture ELISAusing FcγR-expressing K562 cells and a known enhancing antibody 4G2. To verify this method, enhancing activity was measured by NS1 antigen quantitation and traditional enhanced virus titrationsimultaneously, in whichNS1 protein level and infectious viral titer determination showed a linear correlation. Then, ADE activity and neutralizing antibody activities of six primary DENV-1 infected human sera and cross-reactive murine monoclonal antibodies (mAbs) (2D73 and 3E31) were evaluated and analysed by ELISA-ADE and ELISPOT-MNT. It showed 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 by ELISPOT-MNT. The 3E31 mAb showed strong protective neutralization without enhancing viral replication, implying that 3E31 may be as potential immunotherapeutic mAb.Part Ⅲ. Functional properties of DENVEDⅢ-reactive antibodies in human DENV-1-infected sera and rabbit antiserum to EDⅢThe DENV EDIII has been confirmed as target of specific neutralizing antibody, and is also considered to be a promising subunit dengue vaccine candidate. Particularly,purified or recombinant protein with protective non-predominant epitopes could be as subunite vaccine candidates, although several recent studies have shown that anti-EDⅢ antibodies contribute little to the neutralizing or enhancing effect of human DENV-infected serum. Additionally, most research on the role of EDⅢ-reactive antibodies of human convalescence serum focused on DENV-2 or -3 infections, however, more than 60% of dengue patients in southern China belonged to DENV-1 infection. In this study, therefore, neutralization and ADE activity as well as of EDⅢ-binding antibodies titre on homotypic and hetertypic DENVs in human DENV-1-infected sera and rabbit antiserum immunized with recombinant DENV-1 EDⅢ protein were evaluated. Further, the contribution of EDⅢ-reactive antibodies was confirmed by depleting EDⅢ-reactive antibodies from human sera and rabbit antiserum using Dynabeads-EDⅢ conjugates.We performed an analysis of neutralization and ADE activities of EDⅢ-reactive antibodies in both human convalescent sera from patients with primary DENV-1 infection and rabbit antiserum immunized with recombinant DENV-1 EDⅢ protein. The results indicated that serum neutralizationactivity was not associated with titres of EDⅢ-binding antibodies in the human DENV-1-infected sera. The depletion of anti-EDⅢ antibodies from these serum samples revealed that the anti-EDIII antibodies of the patients contributed little to neutralization and ADE. However, the EDIII-reactive antibodies from the rabbit antiserum exhibited protective abilities of neutralization at a high dilution (approximately 1:50,000) and ADE at a low dilution (approximately 1:5,000) for homotypic DENV infection. Notably, the rabbit antiserum showed ADE activity only at a dilution of 1:40 for heterotypic virus infection, implying that EDⅢ-reactive antibodies would be safe in secondary infection with heterotypic viruses. These results suggest that purified or recombinant DENV EDⅢ protein could still be used as a subunit vaccine to provoke an effective and safe antibody response, although DENV EDⅢ is not the predominant antigen of the dengue virus infection process.SummaryIn present work, yeast surface display protein technology and site-directed mutagenesis techniqueswere used to verify that the key amino acid residues of a highly conserved sequence aa310-319 recognized by cross-reactive antibodies is locatedat Q316 and H317 in EDIII protein AB loop. We developed a simple high-throughput ADE assay (ELISA-ADE)for evaluation of enhancing activity of DENV-specific antibodies by quantitative measurement of NS1 antigen in the culture supernatant by NS1 capture ELISA using FcyR-expressing K562 cells and a known enhancing antibody as positive control. The combination of ELISA-ADE and ELISPOT-MNT assays was well used to evaluate of DENV enhancing and neutralizing antibody activities of mAbs and polyclonal antisera. 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.Concerning the effect of EDⅢ-reactive antibody in human infected serum, we found that EDⅢ-reactive antibody ofhuman convalescent with DENV-1 infection also showed a minor in neutralization and enhancement, which may indicate thatnot all of protective epitopes can be as predominant epitope to provoke an effective neutralizing antibody response and even ADE.However, protective non-predominant epitopes may be as purified subunite vaccine candidates to induce a protective response. We prepared anti-EDⅢ rabbit polyclonal antisera by using recombinant DENV-1 EDⅢ proteinas immunogen, and demonstrated thatanti-DENV-1 EDⅢ antibodies plays a key role in neutralizing activity and enhancing activity. Moreover, the EDⅢ rabbit antiserum on heterotypic DENV infection only exhibited a weak ADE activity, indicating the safety in humoral response or potential therapy using antibody.