Specific identification of pathogenic Yersinia enterocolitica by monoclonal antibodies generated against recombinant attachment invasion locus (rAil) protein

Classical pathogenic strains of Yersinia enterocolitica produce a 17 kDa outer membrane protein, Ail (attachment-invasion locus), which mediates bacterial attachment to some cultures epithelial cell lines and invasion of others. In the present study, hybridomas were developed for the production of monoclonal antibodies (MAbs) against Ail protein of Y. enterocolitica. A set of five stabilized hybridoma cell lines were generated, of which, two MAbs, YEA 302 and YEA 303, exhibited specific reaction to the native Ail protein (17 kDa) present in whole cell lysate of Y. enterocolitica strains beside having reaction with rAil. The other three MAbs, YEA 5, 17 and 32, had some cross reactions with proteins other than Ail. Two out of five MAbs were IgG1, two were IgG2b and one in IgM in nature. MAbs (YEA 302 and YEA 303) did not show any cross-reaction with whole cell lysate of Brucella abortus, Vibrio cholerae, Salmonella typhimurium and Escherichia coli and other species of Enterobacteriaceae including Y. pestis in ELISA and Western blot analysis. The presence of Ail protein among the strains recovered from pork and milk samples was evaluated by these sets of MAbs and the results were compared with the duplex PCR. Collectively, the data suggest that these MAbs may have the potential for their use in the detection of pathogenic Y. enterocolitica reliably, rapidly and at a relatively low cost.     

Characterization of Chlamydia trachomatis antigens with monoclonal and polyclonal antibodies

We used monoclonal antibodies (MAbs) to examine the antigenic specificity and biologic function of several Chlamydia trachomatis antigens. Thirteen distinct MAbs to eight C. trachomatis antigens were produced. Six MAbs reacted with unique epitopes on the major outer membrane protein (MOMP) and two of these had neutralizing activity. MAbs were produced to each of the chlamydial antigens with molecular masses of 10, 29, 32, 57, 60, 70, and 75 kilodaltons (kDa). These MAbs showed species and genus specificity in an immunoblot assay. None of the MAbs had neutralizing activity. The epitopes recognized on MOMP, 29-, and 10-kDa (presumably lipopolysaccharide) antigens were surface exposed. MAbs to the 75-kDa, 57-kDa, and MOMP antigens were used for immunoaffinity purification of these antigens to produce monospecific antisera in mice. With polyclonal sera, we found that the 75-kDa antigen was also immunoaccessible and that antibody to MOMP and 75-kDa antigens neutralized C. trachomatis infectivity. We conclude that, in addition to MOMP and lipopolysaccharide, antigens with molecular masses of 75 and 29 kDa are surface exposed. Antibodies to MOMP and 75-kDa antigens can neutralize the organism in vitro.     

A Function of 40 kDa Outer Membrane Protein in Serratia marcescens

The function of one of the outer membrane proteins of Serratia marcescens was investigated. S. marcescens with an abundant 40 kDa outer membrane protein was induced to form spheroplast at a high rate in an isotonic medium in the presence of calcium, although the spheroplasts were generally fragile in the isotonic environment. The degree of spheroplast induction was correlated to the amount of the 40 kDa protein present in the membrane. In the 40 kDa proteinless mutant strains, the spheroplast induction rate was remarkably decreased. Autoradiography of the outer membrane revealed the presence of a calcium-binding protein as a radioactive band whose position coincided with the 40 kDa protein. These results suggest that the 40 kDa protein has an important role in maintaining the structural integrity of the cell wall against osmotic shock.     

Immunological Properties of Borrelia burgdorferi Isolated from the Ixodes ovatus in Shizuoka,Japan

Three strains of spirochetes (IKA1 to 3) were isolated from the midgut of Ixodes ovatus collected in the Ikawa region of the northern part of Shizuoka, Japan. These isolates had eight flagella, and their size and other morphological features were similar to Borrelia burgdorferi. They showed similar motility and reacted with monoclonal antibody (MAb) H9724 against borrelial flagella and with MAb H5332 against the outer surface protein A. These strains showed similar SDS-PAGE profiles to that of B. burgdorferi strain B31 and P/Bi isolated in the U.S.A. and Europe, respectively. Immunoblot with Lyme disease patient serum showed positive reactions with the flagella (41 Kilodalton, kDa), protein C (20 to 22 kDa), and outer surface protein A (29 kDa) of the isolates. Immunological properties, morphological characteristics, and epidemiological features revealed that these isolates were B. burgdorferi.     

Isolation and Characterization of Three Porin-Like Proteins from Vibrio vulnificus: Effect of Different Growth Media on Their Production

The effect of the culture media on the composition of the outer membrane protein of Vibrio vulnificus strain 393 from human blood was examined. Only one major outer membrane protein, with an apparent molecular weight of 37,000 (37K protein) and 34,000 (34K protein), was formed in the cells grown in 3% NaCl-BHI broth and chemically defined medium, respectively. The production of one major outer membrane protein was also observed in other isolates from humans and asari clam when they were grown in 3% NaCl-BHI broth. On the other hand, three major outer membrane proteins, with apparent molecular weights of 48,000 (48K protein), 37,000 (37K protein), and 34,000 (34K protein), were produced in the cells grown in 3% NaCl-nutrient broth. Three proteins, 48K, 37K, and 34K from strain 393, were purified and the amino acid compositions were determined. Although there was a little difference in the composition of amino acid among three proteins, the amino acid compositions of the three porin-like proteins showed characteristic properties of the porins of Escherichia coli and Salmonella typhimurium. Immunoblot analysis of the outer membrane proteins from four vibrios, E. coli, and S. typhimurium using monospecific antisera against these three porin-like proteins showed that only the antiserum against 37K protein cross-reacted with the outer membrane proteins from all the strains tested.     

Characterization of an OmpA-like outer membrane protein of the acidophilic iron-oxidizing bacterium, <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis>

An OmpA family protein (FopA) previously reported as one of the major outer membrane proteins of an acidophilic iron-oxidizing bacterium Acidithiobacillus ferrooxidans was characterized with emphasis on the modification by heat and the interaction with peptidoglycan. A 30-kDa band corresponding to the FopA protein was detected in outer membrane proteins extracted at 75°C or heated to 100°C for 10 min prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, the band was not detected in outer membrane proteins extracted at ≤40°C and without boiling prior to electrophoresis. By Western blot analysis using the polyclonal antibody against the recombinant FopA, FopA was detected as bands with apparent molecular masses of 30 and 90 kDa, suggesting that FopA existed as an oligomeric form in the outer membrane of A. ferrooxidans. Although the fopA gene with a sequence encoding the signal peptide was successfully expressed in the outer membrane of Escherichia coli, the recombinant FopA existed as a monomer in the outer membrane of E. coli. FopA was detected in peptidoglycan-associated proteins from A. ferrooxidans. The recombinant FopA also showed the peptidoglycan-binding activity.     

Monoclonal antibody analysis of neutralization and antibody-dependent enhancement of feline infectious peritonitis virus.

Fifty-four monoclonal antibodies (MAbs) to feline infectious peritonitis virus (FIPV) were characterized according to protein specificity, immunoglobulin subclass, virus neutralization, reactivity with different coronaviruses, and ability to induce antibody-dependent enhancement (ADE) of FIPV infection in vitro. The MAbs were found to be specific for one of three structural proteins of FIPV. A total of 47 MAbs were specific for the 205-kDa spike protein (S), 3 MAbs were specific for the 45-kDa nucleocapsid protein (N), and 4 MAbs were specific for the 26- to 28-kDa membrane protein (M). The S-specific MAbs showed various degrees of cross-reactivity with strains of FIPV, feline enteric coronavirus, canine coronavirus, and porcine transmissible gastroenteritis virus. Nineteen S-specific MAbs neutralized FIPV. A total of 15 of the neutralizing MAbs induced ADE, and all but 1 were of the immunoglobulin G2a subclass. The remaining four neutralizing MAbs that did not induce ADE were of the immunoglobulin G1 subclass. Two S-specific MAbs induced ADE but were nonneutralizing. None of the N- or M-specific MAbs was neutralizing or induced ADE. On the basis of the reactivity patterns of the MAbs with FIPV and related coronaviruses, it was concluded that there is a minimum of five neutralizing sites on S. In most instances, neutralizing MAbs were able to induce ADE, demonstrating a direct relationship between neutralization and enhancement. The difference in immunoglobulin subclass between neutralizing MAbs that induced ADE and those that did not induce ADE suggests that there may be a restriction in the immunoglobulin subclasses capable of mediating ADE.     

Demonstration of a Ferric Vibrioferrin-Binding Protein in the Outer Membrane of Vibrio parahaemolyticus

Under iron-restricted conditions, Vibrio parahaemolyticus produces a siderophore, vibrioferrin, accompanying expression of two major outer membrane proteins of 78 and 83 kDa. Autoradiographic analysis of nondenaturing polyacrylamide gel electrophoregrams of outer membrane preparations previously incubated with [⁵⁵Fe]ferric vibrioferrin revealed a single radiolabeled band, in which the 78-kDa protein was detected predominantly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The antiserum against the purified 78-kDa protein partially inhibited Fe-VF binding to isolated OMPs. The 78-kDa protein was cleaved by the treatment of whole cells with proteinase K, indicating that a portion of this protein is exposed on the surface of the outer membrane. The treated cells lost most of their iron uptake activity mediated by vibrioferrin. These results suggest that the ferric vibrioferrin-binding protein of 78 kDa may function as the receptor for ferric vibrioferrin involved in the initial step of vibrioferrin-mediated iron uptake. Immunoblot analysis using the antiserum against the 78-kDa protein demonstrated that the molecular mass and antigenic properties of the protein were highly conserved among V. parahaemolyticus strains examined. The antiserum also recognized an iron-repressible outer membrane protein of 78 kDa from iron-restricted V. alginolyticus strains, some of which appeared to produce vibrioferrin.     

Monoclonal antibodies against 27.8 kDa protein receptor efficiently block lymphocystis disease virus infection in flounder Paralichthys olivaceus gill cells

In previous research using co-immunoprecipitation, a 27.8 kDa protein in flounder Paralichthys olivaceus gill (FG) cells was found to bind lymphocystis disease virus (LCDV). In this paper, 13 hybridomas secreting monoclonal antibodies (MAbs) against the 27.8 kDa protein were obtained, and 2 MAbs designated as 2G11 and 3D9 were cloned by limiting dilution. Analyzed by indirect enzyme-linked immunosorbent assay (ELISA) and western blotting, the MAbs specifically reacted with the 27.8 kDa protein of FG cells. Confocal fluorescence microscopy and immunogold electron microscopy (IEM) provided evidence that the epitopes recognized by these MAbs were located primarily on the cell membrane and occasionally in the cytoplasm near the cell membrane of FG cells. The MAbs could block LCDV binding after MAbs were pre-incubated with isolated membrane proteins of FG cells in a blocking ELISA, and MAbs also could inhibit LCDV infection of FG cells in culture. Moreover, several target tissues of LCDV in flounder, including gill, stomach, intestine and liver, displayed the presence of the LCDV receptor-27.8 kDa. These results strongly supported the possibility that the 27.8 kDa protein is the putative receptor specific for LCDV infection of FG cells in flounder.     

Infection-enhancing and -neutralizing activities of mouse monoclonal antibodies against dengue type 2 and 4 viruses are controlled by complement levels

Dengue viruses are distributed widely in the tropical and subtropical areas of the world and cause dengue fever and its severer form, dengue hemorrhagic fever. While neutralizing antibodies are considered to play a major role in protection from these diseases, antibody-dependent enhancement (ADE) of infection is an important mechanism involved in disease severity, in addition to the involvement of T lymphocytes. Here, we analyzed relationships between neutralizing and enhancing activities at a clonal level using models of dengue type 2 virus (DENV2) and dengue type 4 virus (DENV4). Totals of 33 monoclonal antibodies (MAbs) against DENV2 and 43 against DENV4 were generated, all directed to the envelope protein. In these MAbs, enhancing activities were shown at subneutralizing doses under normal ADE assay conditions where test samples were heat inactivated. However, the inclusion of commercial rabbit complement or fresh sera from healthy humans in the ADE assay system abolished the enhancing activities of all these MAbs. The reductive effect of fresh sera on enhancing activities was significantly reduced by their heat inactivation or the use of C1q- or C3-depleted sera. In some fresh sera, enhancing activities were shown within a range of 20 to 80% of normal complement levels in a dose-dependent fashion. These results indicate that a single antibody species possesses two distinct activities (neutralizing/enhancing), which are controlled by the level of complement, suggesting the involvement of complement in dengue disease severity. Fresh human sera also tended to reduce enhancing activities more effectively in homologous than heterologous combinations of viruses (DENV2/DENV4) and MAbs (against DENV2/DENV4).     

Surface antigen analysis of group B Neisseria meningitidis outer membrane by monoclonal antibodies: Identification of bactericidal antibodies to class 5 protein

Twenty-four monoclonal antibodies (mAbs) against group B Neisseria meningitidis surface antigens were analyzed by immunoenzymatic assays and by a bactericidal test. Two mAbs were specific to polysaccharide B and one to lipopolysaccharide. The others were directed against outer membrane proteins ranging in molecular mass from 25 to 200 kDa. The outer membrane protein epitopes recognized by the mAbs were not conformational and were located on the outer surface of the microorganism. Linear epitopes on the class 5 protein, exposed on the surface of the membrane, were able to induce bactericidal antibodies to the homologous strain. The susceptibility of Neisseria meningitidis to these antibodies was unchanged when this organism was cultivated under conditions of iron depletion. These results demonstrate that peptides derived from class 5 proteins are potentially important in synthetic peptide or in recombinant protein vaccines containing linear bactericidal epitopes.     

PLCγ1–PKCγ Signaling‐Mediated Hsp90α Plasma Membrane Translocation Facilitates Tumor Metastasis

The 90‐kDa heat shock protein (Hsp90α) has been identified on the surface of cancer cells, and is implicated in tumor invasion and metastasis, suggesting that it is a potentially important target for tumor therapy. However, the regulatory mechanism of Hsp90α plasma membrane translocation during tumor invasion remains poorly understood. Here, we show that Hsp90α plasma membrane expression is selectively upregulated upon epidermal growth factor (EGF) stimulation, which is a process independent of the extracellular matrix. Abrogation of EGF‐mediated activation of phospholipase (PLCγ1) by its siRNA or inhibitor prevents the accumulation of Hsp90α at cell protrusions. Inhibition of the downstream effectors of PLCγ1, including Ca²⁺ and protein kinase C (PKCγ), also blocks the membrane translocation of Hsp90α, while activation of PKCγ leads to increased levels of cell‐surface Hsp90α. Moreover, overexpression of PKCγ increases extracellular vesicle release, on which Hsp90α is present. Furthermore, activation or overexpression of PKCγ promotes tumor cell motility in vitro and tumor metastasis in vivo, whereas a specific neutralizing monoclonal antibody against Hsp90α inhibits such effects, demonstrating that PKCγ‐induced Hsp90α translocation is required for tumor metastasis. Taken together, our study provides a mechanistic basis for the role for the PLCγ1–PKCγ pathway in regulating Hsp90α plasma membrane translocation, which facilitates tumor cell motility and promotes tumor metastasis.     

Investigation on the Fusarium induced stress protein (FISP) in wheat: Immunolocalisation in root cells

Abstract

Fusarium induced-stress-protein (FISP) of ～51 kDa molecular mass was detected in seven day old germinated wheat (Triticum aestivum var Sonalika) seedlings infected with F. oxysporum for a period of seven days. This particular stress protein (FISP) of ～51 kDa was over-expressed in the case of Fusarium infected seedlings compared to the untreated seedlings where the presence of this protein was insignificant. Localisation of this ～51 kDa protein in root tissue by anti-CSAP (Cadmium Stress Associated Protein) antiserum showed a significantly higher number of gold particles in the case of Fusarium infected root tissue compared to the untreated control. A unique type of organised localisation of FISP around the plasma membrane and outer vacuolar membrane suggests its defensive role against Fusarium infection that might be a general stress protein against biotic and abiotic stresses.     

Some aspects of the immune response of koalas (Phascolarctos cinereus) and in vitro neutralization of chlamydia psittaci (koala strains)

Abstract Western-blot analysis was used to study the reaction of koala antisera, two specific polyclonal antibodies and one monoclonal antibody, with chlamydial antigens in koalas infected with Chlamydia psittaci . The koala sera recognized four C. psittaci surface antigens, corresponding to the major outer membrane protein (39.5 kDa), 31 kDa protein, 18 kDa protein and lipopolysaccharide. The S25-23 LPS specific monoclonal antibody inhibited chlamydial infection (55–67%) with both koala strains (type I and type II). Both koala antiserum and rabbit polyclonal antibodies against either type of chlamydia significantly reduced the number of infected cells resulting from type II infections at a dilution of 1 in 20. Rabbit antiserum against type II was effective in neutralizing infection by type II elementary bodies, but was less effective against type I infection. In addition, no koala antiserum was effective in neutralizing type I infection.     

Coordinated expression and immunogenicity of an outer membrane protein from Salmonella enterica serovar Typhi under iron limitation, oxidative stress and anaerobic conditions

Summary Successful pathogens overcome the environmental stresses by the coordinated expression of various genes and eventually proteins. Since, the surface of the microbe is likely to come in contact with the host initially, an attempt was made to identify the outer membrane proteins (OMPs), if any, which may get expressed under more than one environmental conditions simulating the in vivo ones. In the present study, Salmonella enterica serovar Typhi was grown under iron-limited, oxidative stress as well as anaerobic conditions and the OMP profiles were compared. A 69 kDa OMP was found to express with enhanced intensity under the selected stress conditions in comparison to normal conditions. The phenotypic similarity among the proteins was assessed on the basis of their molecular weight, cross reactivity and HPLC. The protein expressed under oxidative stress and anaerobic conditions reacted with the antibodies raised against iron-regulated outer membrane protein (IROMP), indicating the sharing of at least some of the epitopes. A single peak observed after subjecting the pooled 69 kDa protein sample and appearance of a single band on SDS-PAGE thereafter, confirmed the purity and phenotypic similarity of the 69 kDa OMP. Reactivity of pooled 69 kDa protein with 85% of sera from typhoid patients revealed the in vivo expression of this protein. The results of this study indicate the coordination of this phenotype under iron stress, oxidative stress and anaerobic conditions. In view of the expression of the 69 kDa protein under the selected stress conditions and their in vivo immunogenicity, these findings may be relevant for the better understanding of the host–microbe interactions and for the further development of diagnostic and preventive strategies.     

Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

Haemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full-length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N-terminal portion of mature Hap (designated Hap_s). In the present study, we examined the mechanism of processing and secretion of Hap. Site-directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N-terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the C-terminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hap_β) predicted formation of a β-barrel with an N-terminal transmembrane α-helix followed by 14 transmembrane β-strands. Additional analysis revealed that the final β-strand contains an amino acid motif common to other β-barrel outer membrane proteins. Upon deletion of this entire C-terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Hap_s was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C-terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Hap_s. These results provide insight into a growing family of Gram-negative bacterial exoproteins that are secreted by an IgA1 protease-like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of β-barrel proteins to the bacterial outer membrane.     

Cross-Reactivity and Neutralizing Ability of Monoclonal Antibodies against Microcystins

Monoclonal antibodies (MAbs) against the microcystin-leucine-arginine variant (MCYST-LR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. The specificity of the MAbs and their ability to neutralize the toxin were investigated by an indirect enzyme-linked immunosorbent assay (ELISA) and by a neutralizing test in mice, respectively. All MAbs reacted with MCYST-LR and also with the microcystin-arginine-arginine variant (MCYST-RR), 3, 7-didesmethylmicrocystin (MCYST-3, 7-dDMLR) and 7-desmethylmicrocystin (MCYST-7-DMLR). Furthermore, the antibodies reacted with cell-extracts of toxic and non-toxic M. aeruginosa strains. The MAbs can apparently recognize the common configuration, but not the variant-specific structure, in the microcystin molecules. The non-toxic strains apparently contain some substance(s) related antigenically to microcystin. The in vivo toxin-neutralizing ability of MAbs was minimal.     

Protective anti-reovirus monoclonal antibodies and their effects on viral pathogenesis.

We used a recently isolated and characterized panel of monoclonal antibodies (MAbs) specific for cross-reactive determinants on reovirus outer capsid proteins to define mechanisms of antibody-mediated protection in vivo. We studied the capacities of MAbs to protect against lethal infection with reoviruses which differ in site of primary replication, route of spread, and central nervous system tropism. We found the following. (i) MAbs specific for each of the viral outer capsid proteins (sigma 1, sigma 3, and mu 1) and the core spike protein (lambda 2) were protective under certain circumstances. (ii) In vitro properties of MAbs, including isotype, neutralization of viral infectivity, inhibition of virus-induced hemagglutination, and avidity of binding, were poorly predictive of the capacities of MAbs to protect in vivo. (iii) MAbs did not act at a single stage during pathogenesis to mediate protection; instead, protective MAbs were capable of altering a variety of stages in reovirus pathogenesis. (iv) MAbs protective against one reovirus also protected against other reoviruses that utilized different pathogenetic strategies, suggesting that the viral epitope bound by an antibody rather than the pathogenetic strategy employed by the virus is a critical determinant of antibody-mediated protection in vivo. (v) A prominent mechanism of protective MAb action is inhibition of viral spread through nerves from a site of primary replication (e.g., the intestine or muscle tissue) to the central nervous system.     

Azidobenzamido-008, a new photosensitive substrate for the 'multispecific bile acid transporter' of hepatocytes: evidence for a common transport system for bile acids and cyclosomatostatins in basolateral membranes

Cyclo(-Phe(p-NH[1-14C]Ac)-Thr-Lys-(CO(p-N3)C6H4)-Trp-Phe-DPro++ +), in the following named azidobenzamido-008, was synthesized in order to identify binding sites for c(Phe-Thr-Lys-Trp-Phe-DPro), named 008, (a cyclosomatostatin with retro sequence) in liver cell plasma membranes. In the dark the above photolabel was taken up into isolated hepatocytes, inhibiting the sodium dependent uptake of cholate and taurocholate in a competitive manner (Ki for cholate uptake inhibition = 1 microM; Ki for taurocholate uptake inhibition = 5 microM). When activated by flashed light the inhibition became irreversible (IC50 for cholate uptake inhibition = 2 microM; IC50 for taurocholate uptake inhibition = 9 microM) and the activated cyclopeptide bound chiefly to hepatocellular membrane proteins of 67, 54, 50, 37 kDa. Excess of the initial 008, or of cholate or phalloidin partially protected the above membrane components against labeling with 14C-labeled azidobenzamido-008. In contrast AS 30 D ascites hepatoma cells, known to be deficient in bile acid and cyclosomatostatin transport, could not be specifically labeled by azidobenzamido-008. The membrane proteins preferentially labeled in hepatocytes (50 and 54 kDa) are integral glycoproteins. The 67 kDa protein is a hydrophilic nonglycosylated membrane component. Independent of labeling with 14C-labeled azidobenzamido-008 or with 14C-labeled azidobenzamido-taurocholate, the main radioactive peaks in the pH region of 7, 5.5, 5.25 were identical after solubilization with Nonidet P-40 and subsequent isoelectric focusing. Proteins of 67, 54, 50 and 37 kDa could be enriched by use of 008-containing gels in affinity electrophoresis. Binding sites for 008 were not destroyed by SDS or Nonidet P-40 treatment of plasma membranes.     

Functional Analysis of Antibodies against Dengue Virus Type 4 Reveals Strain-Dependent Epitope Exposure That Impacts Neutralization and Protection

Although prior studies have characterized the neutralizing activities of monoclonal antibodies (MAbs) against dengue virus (DENV) serotypes 1, 2, and 3 (DENV-1, DENV-2, and DENV-3), few reports have assessed the activity of MAbs against DENV-4. Here, we evaluated the inhibitory activity of 81 new mouse anti-DENV-4 MAbs. We observed strain- and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domain II (DII) and DIII of the envelope (E) protein. Several anti-DENV-4 MAbs inefficiently inhibited at least one strain and/or genotype, suggesting that the exposure or sequence of neutralizing epitopes varies within isolates of this serotype. Remarkably, flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after preincubation at 37°C. However, increasing the time of preincubation at 37°C or raising the temperature to 40°C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains. Prophylaxis studies in two new DENV-4 mouse models showed that neutralization titers of MAbs after preincubation at 37°C correlated with activity in vivo. Our studies establish the complexity of MAb recognition against DENV-4 and suggest that differences in epitope exposure relative to other DENV serotypes affect antibody neutralization and protective activity.