Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development

Dengue fever is a mosquito (Aedes aegypti) ‐transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus (DENV 1‐4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia (CYD‐TDV), is available after many decades’ efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoural and cellular immunity via both the mucosal and humoral immune systems. Here, we report the production of tetravalent EDIII antigen (EDIII‐1‐4) in stably transformed lettuce chloroplasts. Transplastomic EDIII‐1‐4‐expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII‐1‐4 antigens was demonstrated by immunoblotting, with the EDIII‐1‐4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII‐1‐4. Our in vitro gastrointestinal digestion analysis revealed that EDIII‐1‐4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.     

Efficient Assembly and Secretion of Recombinant Subviral Particles of the Four Dengue Serotypes Using Native prM and E Proteins

Background

Flavivirus infected cells produce infectious virions and subviral particles, both of which are formed by the assembly of prM and E envelope proteins and are believed to undergo the same maturation process. Dengue recombinant subviral particles have been produced in cell cultures with either modified or chimeric proteins but not using the native forms of prM and E.

Methodology/Principal Findings

We have used a codon optimization strategy to obtain an efficient expression of native viral proteins and production of recombinant subviral particles (RSPs) for all four dengue virus (DV) serotypes. A stable HeLa cell line expressing DV1 prME was established (HeLa-prME) and RSPs were analyzed by immunofluorescence and transmission electron microscopy. We found that E protein is mainly present in the endoplasmic reticulum (ER) where assembly of RSPs could be observed. Biochemical characterization of DV1 RSPs secretion revealed both prM protein cleavage and homodimerization of E proteins before their release into the supernatant, indicating that RSPs undergo a similar maturation process as dengue virus. Pulse chase experiment showed that 8 hours are required for the secretion of DV1 RSPs. We have used HeLa-prME to develop a semi-quantitative assay and screened a human siRNA library targeting genes involved in membrane trafficking. Knockdown of 23 genes resulted in a significant reduction in DV RSP secretion, whereas for 22 others we observed an increase of RSP levels in cell supernatant.

Conclusions/Significance

Our data describe the efficient production of RSPs containing native prM and E envelope proteins for all dengue serotypes. Dengue RSPs and corresponding producing cell lines are safe and novel tools that can be used in the study of viral egress as well as in the development of vaccine and drugs against dengue virus.     

Low Cost Tuberculosis Vaccine Antigens in Capsules: Expression in Chloroplasts,Bio-Encapsulation,Stability and Functional Evaluation In Vitro

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts.     

Novel AAV-based genetic vaccines encoding truncated dengue virus envelope proteins elicit humoral immune responses in mice

The envelope protein of dengue virus is involved in host cell attachment for entry and induction of protective immunity. Current efforts are focused on producing a tetravalent vaccine by mixing four monovalent vaccine components. In this work, we developed a genetic vaccine based on a novel adeno-associated viral (AAV) vector expressing the carboxy-terminal truncated envelope protein (79E) of dengue virus. The expression of the recombinant 79E protein in HEK 293 cells was confirmed by Western blot. Vectors packaged with novel AAV capsids (AAV2/8 or AAV2/rh32.33) were injected into C57BL/6 mice intramuscularly. Dengue virus antigen was produced in the mice and induced long-lasting antibody responses against the dengue virus still detectable 20 weeks after immunization. AAV2/8 vaccine induced higher anti-dengue virus antibody levels than AAV2/rh32.33 vaccine or AAV plasmid. Furthermore, the anti-dengue antibodies could neutralize homogeneous dengue virus. These results demonstrated that the AAV vaccines possessed appropriate immunogenicity and could be used for the development of an effective dengue vaccine.     

C-Terminal Helical Domains of Dengue Virus Type 4 E Protein Affect the Expression/Stability of prM Protein and Conformation of prM and E Proteins

Background

The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines.

Methodology/Principal Findings

In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested.

Conclusions/Significance

A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response.     

A tetravalent dengue vaccine based on a complex adenovirus vector provides significant protection in rhesus monkeys against all four serotypes of dengue virus

Nearly a third of the human population is at risk of infection with the four serotypes of dengue viruses, and it is estimated that more than 100 million infections occur each year. A licensed vaccine for dengue viruses has become a global health priority. A major challenge to developing a dengue vaccine is the necessity to produce fairly uniform protective immune responses to all four dengue virus serotypes. We have developed two bivalent dengue virus vaccines, using a complex adenovirus vector, by incorporating the genes expressing premembrane (prM) and envelope (E) proteins of dengue virus types 1 and 2 (dengue-1 and -2, respectively) (CAdVax-Den12) or dengue-3 and -4 (CAdVax-Den34). Rhesus macaques were vaccinated by intramuscular inoculation of a tetravalent dengue vaccine formulated by combining the two bivalent vaccine constructs. Vaccinated animals produced high-titer antibodies that neutralized all four serotypes of dengue viruses in vitro. The ability of the vaccine to induce rapid, as well as sustained, protective immune responses was examined with two separate live-virus challenges administered at 4 and 24 weeks after the final vaccination. For both of these virus challenge studies, significant protection from viremia was demonstrated for all four dengue virus serotypes in vaccinated animals. Viremia from dengue-1 and dengue-3 challenges was completely blocked, whereas viremia from dengue-2 and dengue-4 was significantly reduced, as well as delayed, compared to that of control-vaccinated animals. These results demonstrate that the tetravalent dengue vaccine formulation provides significant protection in rhesus macaques against challenge with all four dengue virus serotypes.     

A human PrM antibody that recognizes a novel cryptic epitope on dengue E glycoprotein

Dengue virus (DENV) is a major mosquito-borne pathogen infecting up to 100 million people each year; so far no effective treatment or vaccines are available. Recently, highly cross-reactive and infection-enhancing pre-membrane (prM)-specific antibodies were found to dominate the anti-DENV immune response in humans, raising concern over vaccine candidates that contain native dengue prM sequences. In this study, we have isolated a broadly cross-reactive prM-specific antibody, D29, during a screen with a non-immunized human Fab-phage library against the four serotypes of DENV. The antibody is capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV) in FcγR-bearing K562 cells. Remarkably, D29 also cross-reacted with a cryptic epitope on the envelope (E) protein located to the DI/DII junction as evidenced by site-directed mutagenesis. This cryptic epitope, while inaccessible to antibody binding in a native virus particle, may become exposed if E is not properly folded. These findings suggest that generation of anti-prM antibodies that enhance DENV infection may not be completely avoided even with immunization strategies employing E protein alone or subunits of E proteins.     

Synthesis and assembly of dengue virus envelope protein fused to cholera toxin B subunit into biologically active oligomers in transgenic tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>)

Dengue is the fastest growing mosquito-borne disease worldwide, causing nearly 400 million infections annually. A universally applicable dengue virus vaccine is required to arrest its spread. Here, we generated an edible dengue vaccine by expressing the dengue fusion protein in tomatoes, which is a desirable expression system owing to the inherent adjuvanticity of alpha tomatine and immunogenicity of the tomato lectin/microbial antigen complex. The B subunit of Vibrio cholera toxin (CTB) was genetically fused to dengue envelope antigen for improved delivery to antigen-presenting cells and enhanced immunogenicity, while avoiding immunological tolerance. We utilized domain III of the dengue envelope protein (EDIII), as it has been shown to induce serotype-specific neutralizing antibodies. The CTB–EDIII fusion gene construct containing an endoplasmic reticulum target sequence was introduced into tomato plants by Agrobacterium tumefaciens-mediated gene transformation, and the expression of CTB–EDIII in transgenic plants was confirmed by DNA, RNA and protein analyses. Accumulated fusion protein accounted for up to 0.015 % of total soluble protein, and it assembled into fully functional pentamers as demonstrated by binding to GM1 ganglioside. Future work will involve testing of transgenic tomatoes for immunogenicity in mice following oral delivery.     

Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is closely related to West Nile (WN), yellow fever (YF), and dengue (DEN) viruses. Its plus-strand genomic RNA carries a single open reading frame encoding a polyprotein that is cleaved into three structural (C, prM/M, and E) and at least seven nonstructural (NS1/NS1'', NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins, based on previous work with WNV, YFV, and DENV. Here, we aimed to profile experimentally all the viral proteins found in JEV-infected cells. We generated a collection of 15 JEV-specific polyclonal antisera covering all parts of the viral protein-coding regions, by immunizing rabbits with 14 bacterially expressed glutathione-S-transferase fusion proteins (for all nine viral proteins except NS2B) or with a chemically synthesized oligopeptide (for NS2B). In total lysates of JEV-infected BHK-21 cells, immunoblotting with these antisera revealed: (i) three mature structural proteins (~12-kDa C, ~8-kDa M, and ~53-kDa E), a precursor of M (~24-kDa prM) and three other M-related proteins (~10-14 kDa); (ii) the predicted ~45-kDa NS1 and its frameshift product, ~58-kDa NS1'', with no evidence of the predicted ~25-kDa NS2A; (iii) the predicted but hardly detectable ~14-kDa NS2B and an unexpected but predominant ~12-kDa NS2B-related protein; (iv) the predicted ~69-kDa NS3 plus two major cleavage products (~34-kDa NS3^N-term and ~35-kDa NS3^C-term), together with at least nine minor proteins of ~16-52 kDa; (v) the predicted ~14-kDa NS4A; (vi) two NS4B-related proteins (~27-kDa NS4B and ~25-kDa NS4B''); and (vii) the predicted ~103-kDa NS5 plus at least three other NS5-related proteins (~15 kDa, ~27 kDa, and ~90 kDa). Combining these data with confocal microscopic imaging of the proteins’ intracellular localization, our study is the first to provide a solid foundation for the study of JEV gene expression, which is crucial for elucidating the regulatory mechanisms of JEV genome replication and pathobiology.     

Pathogen-derived resistance to dengue type 2 virus in mosquito cells by expression of the premembrane coding region of the viral genome.

The full-length premembrane (prM) coding region of the dengue virus type 2 (DEN-2; Jamaica) genome was expressed in C6/36 (Aedes albopictus) cells in either the sense or the antisense orientation from a double subgenomic Sindbis (dsSIN) virus. Northern (RNA) blot analysis confirmed the expression of sense or antisense DEN-2 prM RNA in infected C6/36 cells. PrM protein was demonstrated in cells infected with dsSIN virus expressing DEN-2 sense RNAs by an immunofluorescence assay. C6/36 cells were infected with each dsSIN virus at a multiplicity of infection (MOI) of 50 and challenged 48 h later with DEN-2 virus at an MOI of 0.1. Whereas C6/36 cells infected with a control of dsSIN virus supported high levels of DEN-2 replication, C6/36 cells infected with the dsSIN virus expressing prM antisense RNA were completely resistant to DEN-2 challenge. Cells expressing prM protein or untranslatable prM sense RNA also were resistant to DEN-2 challenge. Cells expressing prM protein demonstrated some breakthrough of DEN-2 virus when challenged at an MOI of 10. However, expressed untranslatable sense prM RNA conferred complete protection to challenge at the high MOI.     

Structures of immature flavivirus particles

Structures of prM-containing dengue and yellow fever virus particles were determined to 16 and 25 A resolution, respectively, by cryoelectron microscopy and image reconstruction techniques. The closely similar structures show 60 icosahedrally organized trimeric spikes on the particle surface. Each spike consists of three prM:E heterodimers, where E is an envelope glycoprotein and prM is the precursor to the membrane protein M. The pre-peptide components of the prM proteins in each spike cover the fusion peptides at the distal ends of the E glycoproteins in a manner similar to the organization of the glycoproteins in the alphavirus spikes. Each heterodimer is associated with an E and a prM transmembrane density. These transmembrane densities represent either an EE or prMprM antiparallel coiled coil by which each protein spans the membrane twice, leaving the C-terminus of each protein on the exterior of the viral membrane, consistent with the predicted membrane-spanning domains of the unprocessed polyprotein.     

A molecular evaluation of dengue virus pathogenesis and its latest vaccine strategies

More than one third of the world’s population living in tropical and subtropical areas of the world is at risk of dengue infections and as many as 100 million people are yearly infected. This disease has reemerged during the past 20 years in the form of an epidemic. Dengue is caused by one of four related serotypes of dengue virus and often leads to severe forms of the disease, resulting commonly from secondary infections. Dengue virus is a mosquito borne virus, belongs to the family Flaviviridae and consists of a single stranded positive sense RNA genome. Like other RNA viruses it escapes defense mechanisms and neutralization attempts by mutations, which make it more resistant and adaptable to its environment. Antiviral strategies and vaccine development is thus impaired and hence to date there is no licensed vaccine available for dengue virus. Here we discuss various efforts made towards the identification of potential vaccine targets for dengue as well as various strategies employed by research groups/pharmaceutical companies towards the development of a successful dengue vaccine.     

Evaluation of chimeric DNA vaccines consisting of premembrane and envelope genes of Japanese encephalitis and dengue viruses as a strategy for reducing induction of dengue virus infection‐enhancing antibody response

Neutralizing antibodies induced by dengue virus (DENV) infection show viral infection‐enhancing activities at sub‐neutralizing doses. On the other hand, preimmunity against Japanese encephalitis virus (JEV), a congener of DENV, does not increase the severity of DENV infection. Several studies have demonstrated that neutralizing epitopes in the genus Flavivirus are mainly located in domain III (DIII) of the envelope (E) protein. In this study, chimeric premembrane and envelope (prM‐E) gene‐based expression plasmids of JEV and DENV1 with DIII substitution of each virus were constructed for use as DNA vaccines and their immunogenicity evaluated. Sera from C3H/He and ICR mice immunized with a chimeric gene containing DENV1 DIII on a JEV prM‐E gene backbone showed high neutralizing antibody titers with less DENV infection‐enhancing activity. Our results confirm the applicability of this approach as a new dengue vaccine development strategy.     

Dengue virus tetra-epitope peptide expressed in lettuce chloroplasts for potential use in dengue diagnosis

Dengue virus causes about 100 million cases of dengue disease per year in the world. Laboratory diagnosis is done mainly by serological techniques, which in many cases use crude virus extracts that may cause cross-reactions to other flaviviruses. These undesirable cross-reactions can be reduced or eliminated by using recombinant proteins based on restricted epitopes. Aiming to decrease flaviviral cross-reactions and non-specific interactions in dengue serological assays, a plant expression system was chosen for recombinant antigen production as a reliable and inexpensive dengue diagnostic tool. In the present report, the lettuce plastid transformation system was applied to achieve efficient and stable tetra-epitope peptide antigen production, and its reactivity was evaluated. For this purpose, one putative epitope at positions 34 to 57 of E protein within the junction site of domains I and II of dengue virus (DENV) 1 to 4 serotypes linked by glycine linkers was expressed in lettuce chloroplasts. The potential immunoreactivity for the four DENV serotypes was evaluated using sera from patients of positive and negative dengue cases. Results indicated an overall sensitivity of 71.7 % and specificity of 100 %. No cross-reactions with the sera of yellow fever-positive or healthy individuals vaccinated against yellow fever were observed. This novel approach may provide an alternative system for the large-scale production of dengue recombinant antigens useful for serodiagnosis.     

Role of Humoral versus Cellular Responses Induced by a Protective Dengue Vaccine Candidate

With 2.5 billion people at risk, dengue is a major emerging disease threat and an escalating public health problem worldwide. Dengue virus causes disease ranging from a self-limiting febrile illness (dengue fever) to the potentially fatal dengue hemorrhagic fever/dengue shock syndrome. Severe dengue disease is associated with sub-protective levels of antibody, which exacerbate disease upon re-infection. A dengue vaccine should generate protective immunity without increasing severity of disease. To date, the determinants of vaccine-mediated protection against dengue remain unclear, and additional correlates of protection are urgently needed. Here, mice were immunized with viral replicon particles expressing the dengue envelope protein ectodomain to assess the relative contribution of humoral versus cellular immunity to protection. Vaccination with viral replicon particles provided robust protection against dengue challenge. Vaccine-induced humoral responses had the potential to either protect from or exacerbate dengue disease upon challenge, whereas cellular immune responses were beneficial. This study explores the immunological basis of protection induced by a dengue vaccine and suggests that a safe and efficient vaccine against dengue should trigger both arms of the immune system.     

表达登革病毒prM基因小干扰RNA的Vero细胞系的建立

prM蛋白是登革病毒膜蛋白M的前体,膜蛋白M对病毒的组装与成熟有重要作用,针对prM基因设计的小干扰RNA（siRNA）可短期抑制登革病毒复制.为了达到长期抑制登革病毒的效果,本研究构建了插入prM siRNA序列的重组慢病毒,利用流式细胞术分选以及杀稻瘟霉素抗性,筛选出稳定表达prM siRNA的非洲绿猴肾细胞（Vero细胞）系.经逆转录PCR及测序验证siRNA序列表达正确. Vero细胞中prM siRNA的表达率约为976%.当受到登革病毒攻击时,表达prM siRNA的Vero细胞能够明显抑制登革病毒prM基因的表达,并抑制登革病毒在Vero细胞中的复制.建立的Vero细胞系可用于RNA干扰防治登革病毒感染的进一步应用研究.     

Recent progress in dengue vaccine development

Dengue virus(DENV) has four distinct serotypes. DENV infection can result in classic dengue fever and life-threatening dengue hemorrhagic fever/dengue shock syndrome. In recent decades, DENV infection has become an important public health concern in epidemic-prone areas. Vaccination is the most effective measure to prevent and control viral infections. However, several challenges impede the development of effective DENV vaccines, such as the lack of suitable animal models and the antibody-dependent enhancement phenomenon. Although no licensed DENV vaccine is available, significant progress has been made. This review summarizes candidate DENV vaccines from recent investigations.     

Yellow fever virus/dengue-2 virus and yellow fever virus/dengue-4 virus chimeras: biological characterization,immunogenicity, and protection against dengue encephalitis in the mouse model

Two yellow fever virus (YFV)/dengue virus chimeras which encode the prM and E proteins of either dengue virus serotype 2 (dengue-2 virus) or dengue-4 virus within the genome of the YFV 17D strain (YF5.2iv infectious clone) were constructed and characterized for their properties in cell culture and as experimental vaccines in mice. The prM and E proteins appeared to be properly processed and glycosylated, and in plaque reduction neutralization tests and other assays of antigenic specificity, the E proteins exhibited profiles which resembled those of the homologous dengue virus serotypes. Both chimeric viruses replicated in cell lines of vertebrate and mosquito origin to levels comparable to those of homologous dengue viruses but less efficiently than the YF5.2iv parent. YFV/dengue-4 virus, but not YFV/dengue-2 virus, was neurovirulent for 3-week-old mice by intracerebral inoculation; however, both viruses were attenuated when administered by the intraperitoneal route in mice of that age. Single-dose inoculation of either chimeric virus at a dose of 10(5) PFU by the intraperitoneal route induced detectable levels of neutralizing antibodies against the homologous dengue virus strains. Mice which had been immunized in this manner were fully protected from challenge with homologous neurovirulent dengue viruses by intracerebral inoculation compared to unimmunized mice. Protection was associated with significant increases in geometric mean titers of neutralizing antibody compared to those for unimmunized mice. These data indicate that YFV/dengue virus chimeras elicit antibodies which represent protective memory responses in the mouse model of dengue encephalitis. The levels of neurovirulence and immunogenicity of the chimeric viruses in mice correlate with the degree of adaptation of the dengue virus strain to mice. This study supports ongoing investigations concerning the use of this technology for development of a live attenuated viral vaccine against dengue viruses.     

Identification of two surface proteins from C6/36 cells that bind dengue type 4 virus.

Dengue viruses infect cells by attaching to a surface receptor, probably through the envelope (E) glycoprotein, located on the surface of the viral membrane. However, the identity of the dengue virus receptor in the mosquito and in mammalian host cells remains unknown. To identify and characterize the molecules responsible for binding dengue virus, overlay protein blot and binding assays were performed with labeled virus. Two glycoproteins of 40 and 45 kDa located on the surface of C6/36 cells bound dengue type 4 virus. Virus binding by total and membrane proteins obtained from trypsin-treated cells was inhibited, while neuraminidase treatment did not inhibit binding. Periodate treatment of cell proteins did not reduce virus binding, but it modified the molecular weight of the polypeptide detected by overlay assays. Preincubation of C6/36 cells with electroeluted 40- and 45-kDa proteins or with specific antibodies raised against these proteins inhibited virus binding. These results strongly suggest that the 40- and 45-kDa surface proteins are putative receptors or part of a receptor complex for dengue virus.