Purification and characterization of verocytotoxin 2

Abstract Five hybrid cell lines secreting monoclonal antibodies (MAbs; LOSM5, 7, 8, 10 and 11) against Streptococcus mutans serotype f 74-kDa saliva receptor (SR) were generated by fusing rats IR983F myeloma cells with splenocytes from rats immunized with affinity purified 74-kDa SR. All the five MAbs recognized both native and denatured forms of the SR. Three partially different epitopes could be delineated on protein 74-kDa by using unlabeled and alkaline phosphatase-labeled MAbs in competitive enzyme-linked immunosorbent assay (ELISA). Two of them are involved in the binding of salivary glycoproteins to S. mutans cells; as demonstrated by the inhibition of saliva binding to S. mutans cells by the MAbs LOSM7, 8 and 11. The five MAbs also reacted with 150-kDa protein, a higher M _r protein and peptide mapping of 150-kDa and 74-kDa SR showed identical patterns for both polypeptides.     

Affinity purification and characterization of salivary components interacting with Streptococcus mutans serotype f 74K SR cell wall protein

Abstract Two salivary components which specifically bind to Streptococcus mutans OMZ 175 (serotype f) 74K SR cell surface protein were purified from human whole saliva, free of immunoglobulins, by using affinity chromatography. Both components were eluted from the column in active monomeric forms having M _r of 75 and 60 K. The two binding components were identified by WB analysis as free secretory component (SC) (75K) and as a 60K protein antigenically related to serum albumin (HSA).     

Complete nucleotide sequence of the sr gene from Streptococcus mutans OMZ 175

The nucleotide sequence encoding the SR protein of Streptococcus mutans OMZ 175 (serotype f) has been determined. The sr gene consists of 4667 bp and codes for a 171177 Da protein. Comparison of the inferred amino acid sequence with the one of PAc antigen from S. mutans MT 8148 (serotype c) indicates a 88% conservation of amino acid residues which reflects the close relatedness of both proteins. Major differences in amino acid composition are located at the C-terminal part of the sequence where only 298 amino acids of the terminal 420 are conserved.     

Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.

We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and Salmonella typhimurium strains. Western immunoblots showed complete immunological cross-reactivity between E. coli B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coli B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli O8:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S. typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact O antigen, as in E. coli O8:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an O antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfa E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies.     

Coaggregation of Porphyromonas gingivalis and Fusobacterium nucleatum PK 1594 is mediated by capsular polysaccharide and lipopolysaccharide

Previous reports have shown that coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum, two important periodontopathogens, is mediated by a galactoside on the surface of P. gingivalis and a lectin on F. nucleatum. In the present study, purified capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of P. gingivalis PK 1924 (serotype K5) were found to be able to bind to F. nucleatum cells and to inhibit binding of F. nucleatum to P. gingivalis serotype K5. Sugar binding studies showed that the requirements for binding of P. gingivalis serotype K5 CPS and LPS to the F. nucleatum lectin are: the presence of a metal divalent ion, an axial free hydroxyl group at position 4 and free equatorial hydroxyl groups at position 3 and 6 of d-galactose. These data suggest that P. gingivalis serotype K5- CPS and LPS act as receptors mediating coaggregation between P. gingivalis and fusobacteria.     

Characterization of an O-antigen bacteriophage from Aeromonas hydrophila.

A unique bacteriophage of Aeromonas hydrophila serotype O:34 was isolated, purified, and characterized. The bacterial surface receptor was shown to be the O-antigen polysaccharide component of lipopolysaccharide specific to serotype O:34, which was chemically characterized. The high molecular weight lipopolysaccharide fraction (a fraction enriched in O antigen) was fully able to inactivate bacteriophage PM1. Phage-resistant mutants of A. hydrophila O:34 were isolated and found to be specifically devoid of lipopolysaccharide O antigen. No other cell-surface molecules were involved in phage binding. The host range of bacteriophage PM1 was found to be very narrow, producing plaques only on A. hydrophila strains from serotype O:34.     

Molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.     

An epitope of the Semliki Forest virus fusion protein exposed during virus-membrane fusion

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.     

Functional and Structural Characterization of Neutralizing Epitopes of Measles Virus Hemagglutinin Protein

Effective vaccination programs have dramatically reduced the number of measles-related deaths globally. Although all the available data suggest that measles eradication is biologically feasible, a structural and biochemical basis for the single serotype nature of measles virus (MV) remains to be provided. The hemagglutinin (H) protein, which binds to two discrete proteinaceous receptors, is the major neutralizing target. Monoclonal antibodies (MAbs) recognizing distinct epitopes on the H protein were characterized using recombinant MVs encoding the H gene from different MV genotypes. The effects of various mutations on neutralization by MAbs and virus fitness were also analyzed, identifying the location of five epitopes on the H protein structure. Our data in the present study demonstrated that the H protein of MV possesses at least two conserved effective neutralizing epitopes. One, which is a previously recognized epitope, is located near the receptor-binding site (RBS), and thus MAbs that recognize this epitope blocked the receptor binding of the H protein, whereas the other epitope is located at the position distant from the RBS. Thus, a MAb that recognizes this epitope did not inhibit the receptor binding of the H protein, rather interfered with the hemagglutinin-fusion (H-F) interaction. This epitope was suggested to play a key role for formation of a higher order of an H-F protein oligomeric structure. Our data also identified one nonconserved effective neutralizing epitope. The epitope has been masked by an N-linked sugar modification in some genotype MV strains. These data would contribute to our understanding of the antigenicity of MV and support the global elimination program of measles.     

Chimpanzee-human monoclonal antibodies for treatment of chronic poliovirus excretors and emergency postexposure prophylaxis

Six poliovirus-neutralizing Fabs were recovered from a combinatorial Fab phage display library constructed from bone marrow-derived lymphocytes of immunized chimpanzees. The chimeric chimpanzee-human full-length IgGs (hereinafter called monoclonal antibodies [MAbs]) were generated by combining a chimpanzee IgG light chain and a variable domain of heavy chain with a human constant Fc region. The six MAbs neutralized vaccine strains and virulent strains of poliovirus. Five MAbs were serotype specific, while one MAb cross-neutralized serotypes 1 and 2. Epitope mapping performed by selecting and sequencing antibody-resistant viral variants indicated that the cross-neutralizing MAb bound between antigenic sites 1 and 2, thereby covering the canyon region containing the receptor-binding site. Another serotype 1-specific MAb recognized a region located between antigenic sites 2 and 3 that included parts of capsid proteins VP1 and VP3. Both serotype 2-specific antibodies recognized antigenic site 1. No escape mutants to serotype 3-specific MAbs could be generated. The administration of a serotype 1-specific MAb to transgenic mice susceptible to poliovirus at a dose of 5 μg/mouse completely protected them from paralysis after challenge with a lethal dose of wild-type poliovirus. Moreover, MAb injection 6 or 12 h after virus infection provided significant protection. The MAbs described here could be tested in clinical trials to determine whether they might be useful for treatment of immunocompromised chronic virus excretors and for emergency protection of contacts of a paralytic poliomyelitis case.     

Characterization of human serotype 1 astrovirus-neutralizing epitopes.

Astroviruses are important agents of pediatric gastroenteritis. To better understand astrovirus antigenic structure and the basis of protective immunity, monoclonal antibodies (MAbs) were produced against serotype 1 human astrovirus. Four MAbs were generated. One MAb (8G4) was nonneutralizing but reacted to all seven serotypes of astrovirus by enzyme-linked immunosorbentassay (ELISA) and immunoperoxidase staining of infected cells. Three MAbs were found to have potent neutralizing activity against astrovirus. The first (5B7) was serotype 1 specific, another (7C2) neutralized all seven human astrovirus serotypes, while the third (3B2) neutralized serotypes 1 and 7. Immunoprecipitation of radiolabeled astrovirus proteins from supernatants of astrovirus-infected cells showed that all three neutralizing antibodies reacted with VP29. MAb 5B7 also reacted strongly with VP26. A competition ELISA showed that all three neutralizing antibodies competed with each other for binding to purified astrovirus virions, suggesting that their epitopes were topographically in close proximity. None of the neutralizing MAbs competed with nonneutralizing MAb 8G4. The neutralizing MAbs were used to select antigenic variant astroviruses, which were then studied in neutralization assays. These assays also suggested a close relationship between the respective epitopes. All three neutralizing MAbs were able to prevent attachment of radiolabeled astrovirus particles to human Caco 2 intestinal cell monolayers. Taken together, these data suggest that the astrovirus capsid protein VP29 may be important in viral neutralization, heterotypic immunity, and virus attachment to target cells.     

Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding.

Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates.     

Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.     

Monoclonal antibodies to reovirus reveal structure/function relationships between capsid proteins and genetics of susceptibility to antibody action.

Thirteen newly isolated monoclonal antibodies (MAbs) were used to study relationships between reovirus outer capsid proteins sigma 3, mu 1c, and lambda 2 (core spike) and the cell attachment protein sigma 1. We focused on sigma 1-associated properties of serotype specificity and hemagglutination (HA). Competition between MAbs revealed two surface epitopes on mu 1c that were highly conserved between reovirus serotype 1 Lang (T1L) and serotype 3 Dearing (T3D). There were several differences between T1L and T3D sigma 3 epitope maps. Studies using T1L x T3D reassortants showed that primary sequence differences between T1L and T3D sigma 3 proteins accounted for differences in sigma 3 epitope maps. Four of 12 non-sigma 1 MAbs showed a serotype-associated pattern of binding to 25 reovirus field isolates. Thus, for reovirus field isolates, different sigma 1 proteins are associated with preferred epitopes on other outer capsid proteins. Further evidence for a close structural and functional interrelationship between sigma 3/mu 1c and sigma 1 included (i) inhibition by sigma 3 and mu 1c MAbs of sigma 1-mediated HA, (ii) enhancement of sigma 1-mediated HA by proteolytic cleavage of sigma 3 and mu 1c, and (iii) genetic studies demonstrating that sigma 1 controlled the capacity of sigma 3 MAbs to inhibit HA. These data suggest that (i) epitopes on sigma 3 and mu 1c lie in close proximity to sigma 1 and that MAbs to these epitopes can modulate sigma 1-mediated functions, (ii) these spatial relationships have functional significance, since removal of sigma 3 and/or cleavage of mu 1c to delta can enhance sigma 1 function, (iii) in nature, the sigma 1 protein places selective constraints on the epitope structure of the other capsid proteins, and (iv) viral susceptibility to antibody action can be determined by genes other than that encoding an antibody's epitope.     

The serotype-specific glucose side chain of rhamnose–glucose polysaccharides is essential for adsorption of bacteriophage M102 to Streptococcus mutans

Bacteriophage M102 is a virulent siphophage that propagates in some serotype c Streptococcus mutans strains, but not in S. mutans of serotype e, f or k. The serotype of S. mutans is determined by the glucose side chain of rhamnose–glucose polysaccharide (RGP). Because the first step in the bacteriophage infection process is adsorption of the phage, it was investigated whether the serotype specificity of phage M102 was determined by adsorption. M102 adsorbed to all tested serotype c strains, but not to strains of different serotypes. Streptococcus mutans serotype c mutants defective in the synthesis of the glucose side chain of RGP failed to adsorb phage M102. These results suggest that the glucose side chain of RGP acts as a receptor for phage M102.     

Identification of human parainfluenza virus type 2 (HPIV-2) V protein amino acid residues that reduce binding of V to MDA5 and attenuate HPIV-2 replication in nonhuman primates

Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV-2 virus that expressed the V and P coding sequences from separate mRNAs. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with MDA5, a cytoplasmic sensor of nonself RNA that induces type I IFN. Like WT HPIV-2, these mutants inhibited IFN-β induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit interferon regulatory factor 3 (IRF3) dimerization or IFN-β induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV-2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV-2 growth in vitro. Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.     

Functional and topographical analyses of epitopes on the hemagglutinin (VP4) of the simian rotavirus SA11.

An immunochemical analysis of the hemagglutinin (VP4) of the simian rotavirus SA11 was performed to better understand the structure and function of this molecule. Following immunization of mice with double-shelled virus particles and VP4-enriched fractions from CsCl gradients, a battery of anti-SA11 hybridomas was generated. A total of 13 clones secreting high levels of anti-VP4 monoclonal antibody (MAb) was characterized and compared with two cross-reactive anti-VP4 MAbs generated against heterologous rhesus (RRV) and porcine (OSU) rotavirus strains. These cross-reactive MAbs effectively neutralized SA11 infectivity in vitro. The epitopes recognized by these 15 MAbs were grouped into six antigenic sites on the SA11 hemagglutinin. These sites were identified following analysis of the MAbs by using a simple competitive binding enzyme-linked immunosorbent assay (ELISA) and biological assays. Three of the antigenic sites were involved in neutralization of virus infectivity in vitro. All the MAbs with neutralization activity and two nonneutralizing MAbs were able to inhibit viral hemagglutination of human erythrocytes. Competitive binding ELISA data showed a positive cooperative binding effect with some pairs of the anti-VP4 MAbs, apparently due to a conformational change induced by the binding of the first MAb. Some of the MAbs also bound better to trypsin-treated virus than to non-trypsin-treated virus. A topographic map for VP4 is proposed on the basis of the observed properties of each antigenic site.     

The catalytic subunit of the DNA polymerase of herpes simplex virus type 1 interacts specifically with the C terminus of the UL8 component of the viral helicase-primase complex.

The herpes simplex virus type 1 (HSV-1) UL8 DNA replication protein is a component of a trimeric helicase-primase complex. Sixteen UL8-specific monoclonal antibodies (MAbs) were isolated and characterized. In initial immunoprecipitation experiments, one of these, MAb 804, was shown to coprecipitate POL, the catalytic subunit of the HSV-1 DNA polymerase, from extracts of insect cells infected with recombinant baculoviruses expressing the POL and UL8 proteins. Coprecipitation of POL was dependent on the presence of UL8 protein. Rapid enzyme-linked immunosorbent assays (ELISAs), in which one protein was bound to microtiter wells and binding of the other protein was detected with a UL8- or POL-specific MAb, were developed to investigate further the interaction between the two proteins. When tested in the ELISAs, five of the UL8-specific MAbs consistently inhibited the interaction, raising the possibility that these antibodies act by binding to epitopes at or near a site(s) on UL8 involved in its interaction with POL. The epitopes recognized by four of the inhibitory MAbs were approximately located by using a series of truncated UL8 proteins expressed in mammalian cells. Three of these MAbs recognized an epitope near the C terminus of UL8, which was subjected to fine mapping with a series of overlapping peptides. The C-terminal peptides were then tested in the ELISA for their ability to inhibit the POL-UL8 interaction: the most potent exhibited a 50% inhibitory concentration of approximately 5 microM. Our findings suggest that the UL8 protein may be involved in recruiting HSV-1 DNA polymerase into the viral DNA replication complex and also identify a potential new target for antiviral therapy.     

Characterization and epitope mapping of neutralizing monoclonal antibodies produced by immunization with oligomeric simian immunodeficiency virus envelope protein

In an attempt to generate broadly cross-reactive, neutralizing monoclonal antibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two immunization protocols using different preparations of oligomeric SIV envelope (Env) glycoproteins. In the first protocol, mice were immunized with soluble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzyme-linked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generated. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of Env-expressing cells, and preferentially recognized monomeric Env protein. A subset of these antibodies directed toward the V1/V2 loop, the V3 loop, or nonlinear epitopes were capable of neutralizing CP-MAC, a closely related isolate (SIVmac1A11), and/or two more divergent strains (SIVsmDeltaB670 CL3 and SIVsm543-3E). In the second protocol, mice were immunized with unfixed CP-MAC-infected cells and MAbs were screened for the ability to inhibit cell-cell fusion. In contrast to MAbs generated against sgp140, the seven MAbs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferentially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsmDeltaB670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both groups. MAbs to the V3 loop and one MAb reactive with the V1/V2 loop interfered with CCR5 binding, indicating that these regions of Env play similar roles for SIV and human immunodeficiency virus. Remarkably, several of the MAbs generated against infected cells blocked CCR5 binding in a V3-independent manner, suggesting that they may recognize a region analogous to the conserved coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently than infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.     

Sialylation of Streptococcus suis serotype 2 is essential for capsule expression but is not responsible for the main capsular epitope

The capsular polysaccharide is a critical virulence factor of the swine and zoonotic pathogen Streptococcus suis serotype 2. The capsule of this bacterium is composed of five different sugars, including terminal sialic acid. To evaluate the role of sialic acid in the pathogenesis of the infection, the neuC gene, encoding for an enzyme essential for sialic acid biosynthesis, was inactivated in a highly virulent S. suis serotype 2 strain. Using transmission electron microscopy, it was shown that inactivation of neuC resulted in loss of expression of the whole capsule. Compared to the parent strain, the ΔneuC mutant strain was more phagocytosed by macrophages and was also severely impaired in virulence in a mouse infection model. Both native and desialylated S. suis serotype 2 purified capsular polysaccharides were recognized by a polyclonal anti-whole cell S. suis serotype 2 serum and a monospecific polyclonal anti-capsule serotype 2 serum. In contrast, only the native capsular polysaccharide was recognized by a monoclonal antibody specific for the sialic acid moiety of the serotype 2 capsule. Together, our results infer that sialylation of S. suis serotype 2 may be essential for capsule expression, but that this sugar is not the main epitope of this serotype.