Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries

Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.     

Human monoclonal antibodies specific for conformation-sensitive epitopes of V3 neutralize human immunodeficiency virus type 1 primary isolates from various clades

The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3(JR-CSF) sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.     

Human monoclonal antibodies against surface antigens of Langerhans cells from lymphocytes of diabetics transformed by Epstein-Barr virus

Human monoclonal antibody against islet cell surface antigens was generated from a pre-diabetic patient's peripheral blood lymphocytes transformed with Epstein-Barr virus. Reactivity of these transformed lymphocytes was evaluated using indirect immunofluorescence on rat islet cell suspensions and frozen sections of human pancreas. Several lymphoblastoid cell lines that react with islet cell surface were obtained. Preliminary immunoblots with enriched rat islet cell membrane antigens suggest a reactivity toward a 64 kdalton antigen.     

Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes

The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.     

Oral immunisation of mice with transgenic rice calli expressing cholera toxin B subunit fused to consensus dengue cEDIII antigen induces antibodies to all four dengue serotypes

Dengue virus (DENV) infection is an emerging global health threat. DENV consists of four distinct serotypes, necessitating a tetravalent vaccine. In this study, expression of consensus envelope protein domain III (cEDIII) fused to cholera toxin B subunit (CTB) in transgenic rice calli was improved using the luminal binding protein BiP at the N-terminus and the SEKDEL signal sequences at the C-terminus, targeting the recombinant protein to endoplasmic reticulum (ER). We found that the fusion protein showed higher levels of expression when compared to the fusion proteins using rice amylase 3D (RAmy3D) or CTB native signal sequence only. The CTB-cEDIII fusion protein was evaluated as an oral dengue vaccine candidate in mice. Serotype specific systemic IgG antibodies and specific IgA response in feces were detected and furthermore, T cell proliferation and high frequency antibody-secreting B cells were detected in the spleen. These results suggest the possible use of plant-based dengue tetravalent vaccine targeted to the mucosal immune system for induction of systemic and mucosal immune responses to DENV infection.     

Analysis of human monoclonal antibodies derived from lymphocytes of patients with cancer

Human lymphocytes from tumor-bearing patients and normal individuals have been fused with the NS-1 mouse myeloma line or the LICR -LON- HMY2 ( LICR -2) or SK0 -007 human cell lines. For a given number of lymphocytes, fusions with NS-1 produced 8 times more clones than fusions with LICR -2 and greater than 20 times more clones than fusions with SK0 -007. The percentage of clones that secrete human immunoglobulin (Ig) and the range of Ig production were comparable for clones derived from the three myeloma/lymphoblastoid lines. Clones derived from fusions with LICR -2 and SK0 -007 were found to secrete new species of light and heavy Ig chains in addition to those of the myeloma/lymphoblastoid lines, and clones derived from fusions with NS-1 secreted human Ig and contained both mouse and human chromosomes, which indicates that true hybrid cells were derived from fusions with each of the myeloma/lymphoblastoid lines under study. The stability of Ig production was similar for clones derived from fusions with NS-1, LICR -2, or SK0 -007; these results were comparable to those obtained with standard mouse/mouse hybrids. Stable clones producing human monoclonal antibodies that react with cell surface, cytoplasmic, cytoskeletal, nuclear, or nucleolar antigens have been isolated from tumor-bearing patients and normal individuals. A number of human monoclonal antibodies reactive with cytoskeletal antigens appear to be directed against components of the intermediate filament family. Techniques for the production of human monoclonal antibody appear to be sufficiently advanced to initiate a serological dissection of the humoral immune response to cancer.     

Elevated levels of cell-free circulating DNA in patients with acute dengue virus infection

Background

Apoptosis is thought to play a role in the pathogenesis of severe dengue and the release of cell-free DNA into the circulatory system in several medical conditions. Therefore, we investigated circulating DNA as a potential biomarker for severe dengue.

Methods and Findings

A direct fluorometric degradation assay using PicoGreen was performed to quantify cell-free DNA from patient plasma. Circulating DNA levels were significantly higher in patients with dengue virus infection than with other febrile illnesses and healthy controls. Remarkably, the increase of DNA levels correlated with the severity of dengue. Additionally, multivariate logistic regression analysis showed that circulating DNA levels independently correlated with dengue shock syndrome.

Conclusions

Circulating DNA levels were increased in dengue patients and correlated with dengue severity. Additional studies are required to show the benefits of this biomarker in early dengue diagnosis and for the prognosis of shock complication.     

Protective efficacy of nonneutralizing monoclonal antibodies in acute infection with murine leukemia virus.

We have used an experimental retrovirus infection to study the roles played by different antibodies in resistance to both infection and disease. A molecularly cloned chimeric murine leukemia virus was used to induce acute lethal neurological disease in neonatal mice. A panel of monoclonal antibodies directed against the Gag and Env proteins was tested for protective efficacy. In vitro neutralization assays demonstrated that anti-Env antibodies gave different degrees of neutralization, while no anti-Gag neutralized the virus. In vivo experimental endpoints were onset of clinical signs and premoribund condition. As expected, different anti-Env antibodies demonstrated different degrees of protection which correlated with their neutralizing abilities. Surprisingly, anti-Gag antibodies directed against both p15 (MA protein) and p30 (CA protein) were also protective, significantly delaying the onset of disease. No protection was seen with either of two control antibodies. The protection with anti-Gag was dose related and time dependent and was also produced with Fab fragments. Treatment with anti-Gag did not prevent viremia but resulted in a slight slowing in viremia kinetics and decreased levels of virus in the central nervous systems of mice protected from disease. These data indicate that nonneutralizing antiretroviral antibodies can influence the outcome of retroviral disease. The data also suggest a functional role for cell surface expression of Gag proteins on murine leukemia virus-infected cells.     

Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection

Although maternal human immunodeficiency virus type 1 (HIV-1) transmission occurs during gestation, intrapartum and postpartum (by breast-feeding), 50-70% of all infected children seem to acquire HIV-1 shortly before or during delivery. Epidemiological evidence indicates that mucosal exposure is an important aspect of intrapartum HIV transmission. A simian immunodeficiency virus (SIV) macaque model has been developed that mimics the mucosal exposure that can occur during intrapartum HIV-1 transmission. To develop immunoprophylaxis against intrapartum HIV-1 transmission, we used SHIV-vpu+ (refs. 5,6), a chimeric simian-human virus that encodes the env gene of HIV-IIIB. Several combinations of human monoclonal antibodies against HIV-1 have been identified that neutralize SHIV-vpu+ completely in vitro through synergistic interaction. Here, we treated four pregnant macaques with a triple combination of the human IgG1 monoclonal antibodies F105, 2G12 and 2F5. All four macaques were protected against intravenous SHIV-vpu+ challenge after delivery. The infants received monoclonal antibodies after birth and were challenged orally with SHIV-vpu+ shortly thereafter. We found no evidence of infection in any infant during 6 months of follow-up. This demonstrates that IgG1 monoclonal antibodies protect against mucosal lentivirus challenge in neonates. We conclude that epitopes recognized by the three monoclonal antibodies are important determinants for achieving substantial protection, thus providing a rational basis for AIDS vaccine development.     

Serotyping Coxiella burnetii isolates from acute and chronic Q fever patients by using monoclonal antibodies

Abstract Four mouse monoclonal antibodies reacting with Coxiella burnetii lipopolysaccharide antigens were produced and used in serotyping 17 C. burnetii isolates from acute Q fever and Q fever endocarditis patients in France. Two monoclonal antibodies (1B2 and 3B6) were considered specific for the Priscilla strain, a representative of Q fever endocarditis isolates, and did not react with the Nine Mile strain, which is representative of acute Q fever isolates. Monoclonal antibodies Nos. 1B2 and 3B6 reacted with 75% (3/4) acute Q fever isolates and 85% (11/13) of endocarditis isolates from France. It is reasonable to conclude that Priscilla-like strains cause both acute Q fever and Q fever endocarditis. The hypothesis that Priscilla-like strains only are associated with Q fever endocarditis should be reconsidered.     

Bispecific monoclonal antibodies mediate binding of dengue virus to erythrocytes in a monkey model of passive viremia

Dengue viruses (DEN), causative agents of dengue fever (DF) and more severe dengue hemorrhagic fever (DHF)/dengue shock syndrome, infect over 100 million people every year. Among those infected, up to one-half million people develop DHF, which requires an extensive hospital stay. Recent reports indicate that there is a significant correlation between virus titer in the bloodstream of infected individuals and the severity of the disease, especially the development of DHF. This suggests that if there is a procedure to reduce viremia in infected subjects, then the severity of the disease may be controlled during the critical early stages of the disease before it progresses to DHF. We have generated bispecific mAb complexes (heteropolymer(s), HP), which contain a mAb specific for the DEN envelope glycoprotein cross-linked with a second mAb specific for the primate E complement receptor 1. These HP facilitate rapid binding of DEN to human and monkey E in vitro, with approximately 90% bound within 5 min. Furthermore, in a passive viremia monkey model established by continuous steady state infusion of DEN, injection of HP during the steady state promoted rapid binding of DEN to the E, followed by subsequent clearance from the vascular system. Moreover, HP previously infused into the circulation is capable of efficiently capturing a subsequent challenge dose of DEN and binding it to E. These data suggest that HP potentially can be useful for alleviating DEN infection-associated symptoms by reducing titers of free virus in the vascular system.     

Human serum facilitates hepatitis C virus infection, and neutralizing responses inversely correlate with viral replication kinetics at the acute phase of hepatitis C virus infection

The factors leading to spontaneous clearance of hepatitis C virus (HCV) or to viral persistence are elusive. Understanding virus-host interactions that enable acute HCV clearance is key to the development of more effective therapeutic and prophylactic strategies. Here, using a sensitive neutralization assay based on infectious HCV pseudoparticles (HCVpp), we have studied the kinetics of humoral responses in a cohort of acute-phase patients infected during a single nosocomial outbreak in a hemodialysis center. The 17 patients were monitored for the spontaneous outcome of HCV infection for 6 months before a treatment decision was made. Blood samples were taken frequently (15 +/- 4 per patient). Phylogenetic analysis of the predominant virus(es) revealed infection by only one of two genotype 1b strains. While all patients seroconverted, their sera induced two opposing effects in HCVpp infection assays: inhibition and facilitation. Furthermore, the ability of sera to facilitate or inhibit infection correlated with the presence of either infecting HCV strain and divided the patients into two groups. In group 1, the progressive emergence of a relatively strong neutralizing response correlated with a fluctuating decrease in high initial viremia, leading to control of viral replication. Patients in group 2 failed to reduce viremia within the acute phase, and no neutralizing responses were detected despite seroconversion. Strikingly, sera of group 2, as well as naive sera, facilitated infection by HCVpp displaying HCV glycoproteins from different genotypes and strains, including those retrieved from patients. These results provide new insights into the mechanisms of viral persistence and immune control of viremia.     

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.     

Mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2+ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response.

After intravenous infection of mice, lymphocytic choriomeningitis virus multiplied in spleens and livers, attaining highest concentrations on days 4 to 6. The subsequent clearance was as rapid, and 8 to 10 days after inoculation, infectivity was usually below detectability. During the effector phase of virus elimination, both cytotoxic T-cell (CTL) activity and the number of cells producing antiviral antibodies were high. Monoclonal antibodies directed against T lymphocytes and T-lymphocyte subsets were inoculated once intravenously 5, 6, or 7 days after infection of the animals, and the effects on antiviral immune responses, as well as on elimination of virus from the organs, were determined. Treatment with anti-Thy-1 and anti-Lyt-2 antibodies blocked elimination of the virus and profoundly diminished the activity of spleen CTLs but reduced the antibody response partially (anti-Thy-1) or increased it (anti-Lyt-2). In contrast, treatment with the anti-L3T4 antibody had essentially no effect on either virus elimination or CTL response but abolished antibody production. We conclude that Lyt-2+ (cytotoxic-suppressive) T lymphocytes are needed for elimination of the virus and also regulate the humoral response but that antiviral antibodies are not essential for control of the infection.     

Temporal distribution of dengue virus serotypes in Colombian endemic area and dengue incidence. Re-introduction of dengue-3 associated to mild febrile illness and primary infection

We have investigated the temporal distribution of dengue (DEN) virus serotypes in the department (state) of Santander, Colombia, in relation to dengue incidence, infection pattern, and severity of disease. Viral isolation was attended on a total of 1452 acute serum samples collected each week from 1998 to 2004. The infection pattern was evaluated in 596 laboratory-positive dengue cases using an IgG ELISA, and PRNT test. The dengue incidence was documented by the local health authority. Predominance of DEN-1 in 1998 and DEN-3 re-introduction and predominance in 2001-2003 coincided with outbreaks. Predominance of DEN-2 in 2000-2001 coincided with more dengue hemorrhagic fever (DHF). DEN-4 was isolated in 2000-2001 and 2004 but was not predominant. There was an annual increase of primary dengue infections (from 13.7 to 81.4%) that correlated with frequency of DEN-3 (r = 0.83; P = 0.038). From the total number of primary dengue infections DEN-3 (81.3%) was the most frequent serotype. DHF was more frequent in DEN-2 infected patients than in DEN-3 infected patients: 27.5 vs 10.9% (P < 0.05). DEN-3 viruses belonged to subtype C (restriction site-specific-polymerase chain reaction) like viruses isolated in Sri-Lanka and other countries in the Americas. Our findings show the importance of continuous virological surveillance to identify the risk factors of dengue epidemics and severity.     

Characterization of syngeneic rat monoclonal antibodies to the HSN tumor using syngeneic monoclonal anti-idiotopic antibodies

Twelve syngeneic anti-idiotopic mAb (anti-idiotypic/idiotopic antibodies Ab2)) were prepared from CBH/Cbi rats immunized with one of three monoclonal anti-HSN antibodies (Ab1) (11/160, ALN/11/53, or ALN/16/53) specific for the HSN tumor. The sera of the rats used for hybridoma production and all of the monoclonal Ab2 specifically inhibited the binding to HSN of the immunizing Ab1 only. It is concluded that, in this completely syngeneic system, only the private idiotopes associated with the antibody-combining site were immunogenic. Analyses using Western blotting showed that the Ab2 bound to intact Ab1 and to isolated H chains where the intra-strand disulfide bonds remained intact. The Ab2 did not bind to L chains or to fully reduced H chains of the Ab1. It is concluded that the idiotopes expressed on the H chain were conformational. When a panel of 13 monoclonal Ab1 (including the three used for immunization) were tested for reactivity with the Ab2, three reacted specifically with their respective Ab2 and 8 gave no binding suggesting that each Ab1 had a distinct idiotypic specificity despite the fact that all the Ab1 competed with each other for binding to Ag. However, the two remaining Ab1 (ALN/9/94 and ALN/12/17) generated from different tumor-bearing rats, were found to possess the same idiotypic specificity as 11/160. A detailed analysis using seven Ab2 raised against 11/160 showed that while the idiotype of ALN/9/94 and 11/160 were very similar, that of ALN/12/17 showed some clear differences. These three Ab1 have been shown previously to bind a sequential epitope on the HSN Ag in Western blots and it is postulated that the common idiotype of these Ab1 reflects their recognition of a sequential epitope. This may also account for the relatively frequent occurrence in tumor bearer sera of antibodies with this Id.     

Studies on the antigenic structure of Japanese encephalitis virus using monoclonal antibodies

The antigenic relationships among 11 strains of Japanese encephalitis (JE) virus were analyzed by using monoclonal antibodies (NARMA) against the Nakayama-RFVL strain in hemagglutination-inhibition (HI) and neutralization (Nt) tests. Of the 14 JE virus-specific HI antibodies, all except NARMA 5 showed Nt reactivity with the homologous strain. The HI and Nt titers of these antibodies were not parallel. The 14 antibodies included the following characteristic antibodies: NARMA 3 is a species-specific antibody with HI and Nt reactivities against JE virus, NARMA 13 is a species-specific HI antibody, NARMA 6 is a Nakayama strain-specific antibody with HI and Nt reactivities, and NARMA 5 is a Nakayama strain-specific HI antibody. The 11 strains of JE virus were divided into four major antigenic groups. However, slight antigenic differences were found among some strains of the same group. Furthermore, competitive binding assays were performed to determine the distribution of antigenic determinants by enzyme-linked immunosorbent assay. The results suggest the existence of at least five HI sites on the JE virus virion, and indicate that the JE species-specific HI site and the flavivirus genus-specific HI site are topologically distinct.