Antifungal Activity of a Human Antiglucuronoxylomannan Antibody

The human immunoglobulin M (IgM) monoclonal antibody (MAb) 2E9 binds the glucuronoxylomannan (GXM) of Cryptococcus neoformans serotypes A, B, and D. This study was undertaken to determine the opsonic efficacy of 2E9 and its ability to promote the antifungal activity of human polymorphonuclear neutrophils (PMNs) against C. neoformans. We incubated purified PMNs with fluorescein isothiocyanate-labeled C. neoformans cells that were treated with the GXM IgM 2E9, IgM antibodies that do not bind GXM, and rabbit and human factor-B-deficient serum as complement sources. PMN-associated C. neoformans cells fluoresced and were detected with a fluorescence-activated cell sorter. The amount of phagocytosis was defined as the percent fluorescing PMNs, which was 37% for yeast cells opsonized with 2E9 plus rabbit serum and 57% for yeast cells opsonized with 2E9 plus factor-B-deficient serum. Phagocytosis was significantly greater for yeast cells that were treated with 2E9 plus a complement source than for yeast cells treated with the complement sources alone or treated with the control IgMs alone or with the complement sources. Fluorescence quenching and light and electron microscopy of the phagocytosis mixtures revealed that 2E9-opsonized yeast cells were internalized by PMNs. Maximal inhibition of C. neoformans growth occurred when PMNs were cocultured with yeast cells that were opsonized with 2E9 plus a complement source. Our data demonstrate that the human GXM IgM 2E9 can mediate PMN phagocytosis and C. neoformans growth inhibition in vitro. These findings strongly suggest that antibody-mediated deposition of complement components on the cryptococcal capsule can augment PMN complement receptor-mediated antifungal activity. Antibody activation of complement-mediated effector cell antifungal mechanisms may play a role in host defense against cryptococcosis and represents a goal for the use of MAbs to treat or prevent human C. neoformans infections.     

In vitro and in vivo activity of polyclonal and monoclonal human immunoglobulins G, M, and A against Pseudomonas aeruginosa lipopolysaccharide.

We evaluated the in vitro opsonophagocytic killing activity of monoclonal human immunoglobulin G (IgG), IgM, and IgA specific for Pseudomonas aeruginosa lipopolysaccharide and the in vivo protective capacity in neutropenic mice of both monoclonal and purified polyclonal IgG, IgM, and IgA. Monoclonal IgM was efficacious in mediating opsonophagocytic killing only in conjunction with complement, whereas monoclonal IgG opsonic killing was potentiated by complement, and monoclonal IgA opsonic killing was independent of complement. These findings are similar to those previously reported for purified polyclonal IgM, IgG, and IgA. The monoclonal and polyclonal immunoglobulins had comparable 50% protective doses in neutropenic mice (range, 0.28 to 0.46 microgram per mouse). The protective activity of IgM in neutropenic mice was abolished by cobra venom factor treatment, whereas IgG and IgA maintained efficacy in cobra venom factor-treated mice. These data indicate that all three major human serum immunoglobulin isotypes have opsonophagocytic and protective activities against P. aeruginosa, with a critical role for complement in the function of IgM.     

Monoclonal antibody-based capture enzyme immunoassays for specific serum immunoglobulin G (IgG), IgA, and IgM antibodies to respiratory syncytial virus

Monoclonal antibodies (MAbs) to the fusion protein (F), attachment protein (G), and nucleoprotein (N) of respiratory syncytial (RS) virus were evaluated for use as detector antibodies in immunoglobulin G (IgG), IgA, and IgM capture enzyme immunoassays. MAb assays were tested against assays using polyclonal antibodies (PAbs) with serum specimens from patients with and without evidence of recent RS virus infection. Assays developed with N MAbs were comparable to or better than PAb assays for detecting specific IgG and IgM antibodies but were somewhat less sensitive for IgA. F MAb assays were less sensitive for IgG and IgM antibodies but identified specific IgA in some specimens negative by N MAb assay. G MAb assays were insensitive for IgG and IgM antibodies but did detect about 50% of the IgA antibodies identified by the PAb assay. The basis for the low sensitivity of the G MAb assays is unclear, since many of these specimens were positive for IgG antibodies to G by Western immunoblot. The sensitivity of MAb assays varied with patient age: N MAb assays detected specific antibody responses to RS virus in all immunoglobulin classes in both adults and infants less than 1 year of age, F MAb assays detected specific IgG responses in adults and IgA responses in both adults and infants, and G MAb assays only detected IgA responses in adults. A mixture of N and F MAbs was complementary overall, identifying 54 of 55 (IgG), 51 of 52 (IgA), and 16 of 17 (IgM) serum specimens positive by PAb assay. These MAb assays were also specific with specimens tested from persons without a history of recent RS virus infection. The availability of these MAb-based assays offers other laboratories the opportunity to have long-term, standardized reagents and tests for serological diagnosis of RS virus infection.     

The significance of immunofluorescent immunoglobulin inclusions in polymorphonuclear leucocytes for the detection of circulating immune complexes

Cytoplasmic inclusions of immunoglobulins and complement, detected by fluorescent antibodies in polymorphonuclear leucocytes (PMNs) that have been incubated with sera of certain patients, are considered to represent immune complexes (IC). The usefulness of this test--the indirect PMN phagocytosis test (IPPT)--for the detection of circulating IC was investigated using preparations of free and heat-aggregated immunoglobulins. Free IgG and IgM were phagocytozed by PMNs at high concentrations only, while free IgA was not phagocytozed at all. Normal human serum slightly enhanced the uptake of free IgG and IgM, but not of free IgA. Aggregates of IgG and IgA underwent phagocytosis at low concentrations, but IgM aggregates were not taken up more readily than free IgM. The uptake of IgG aggregates decreased in the presence of serum, while there was no influence upon the phagocytosis of IgA aggregates. Phagocytosis of C3 occurred only with IgG aggregates. In the presence of aggregates of IgG or IgA the phagocytosis of free immunoglobulins of other classes, in particular IgM, increased. The results of the IPPT for patients' sera showed that inclusions of C3 were found more frequently in combination with IgA or IgM than with IgG. Comparison with the 125I-Clq binding assay and the anti-IgA inhibition binding assay disclosed significant correlation between the phagocytosis of IgG and the precipitation of 125I-Clq and between the phagocytosis of IgA and the results of the anti-IgA inhibition binding assay. The PMN phagocytosis test may be useful for the detection of IgG and IgA containing IC but inclusion of IgM and C3 should be interpreted with some reserve.     

A monoclonal antibody to Candida albicans enhances mouse neutrophil candidacidal activity.

A monoclonal antibody (MAb) to Candida albicans (MAb B6.1) that protects against candidiasis and the nonprotective MAb B6 were compared for ability to support neutrophil (polymorphonuclear leukocyte [PMN]) candidacidal activity. Both MAbs are immunoglobulin M, and each recognizes distinct C. albicans mannan cell wall determinants. PMN candidacidal activity was assessed by transmission electron microscopy and by an in vitro killing assay. The results indicated that MAb B6.1, but not MAb B6, enhances ingestion and killing of yeast cells by PMN in the presence of serum complement.     

A peptide mimotope of type 8 pneumococcal capsular polysaccharide induces a protective immune response in mice

Increasing antibiotic resistance and a rising patient population at risk for infection due to impaired immunity underscore the importance of vaccination against pneumococci. However, available capsular polysaccharide vaccines are often poorly immunogenic in patients at risk for pneumococcal disease. The goal of this study was to explore the potential of peptide mimotopes to function as alternative vaccine antigens to elicit a type-specific antibody response to pneumococci. We used a human monoclonal immunoglobulin A (IgA) antibody (NAD) to type 8 Streptococcus pneumoniae capsular polysaccharide (type 8 PS) to screen a phage display library, and the phage PUB1 displaying the peptide FHLPYNHNWFAL was selected after three rounds of biopanning. Inhibition studies with phage-displayed peptide or the peptide PUB1 and type 8 PS showed that PUB1 is a mimetic of type 8 PS. PUB1 conjugated to tetanus toxoid (PUB1-TT) induced a type 8 PS-specific antibody response in BALB/c mice, further defining it as a mimotope of type 8 PS. The administration of immune sera obtained from PUB1-TT-immunized mice earlier (days 14 and 21) and later (days 87 and 100) after primary and reimmunization resulted in a highly significant prolongation of the survival of naive mice after pneumococcal challenge compared to controls. The survival of PUB1-TT-immunized mice was also prolonged after pneumococcal challenge nearly 4 months after primary immunization. The efficacy of PUB1-TT-induced immune sera provides proof of principle that a mimotope-induced antibody response can protect against pneumococci and suggests that peptide mimotopes selected by type-specific human antibodies could hold promise as immunogens for pneumococci.     

Cross-reactive monoclonal antibodies for diagnosis of pneumococcal meningitis.

A diagnostic test for the detection of Streptococcus pneumoniae meningitis was developed using monoclonal antibodies (MAbs) to phosphocholine (PC) and non-PC determinants of pneumococcal teichoic acids. These MAbs do not recognize other bacteria that commonly cause meningitis. By using a dot blot assay, these MAbs were compared with a polyvalent pneumococcal capsular omniserum and an antiserum made to whole cells for their ability to detect pneumococci in infected spinal fluids. An immunoglobulin M (IgM) anti-PC antibody gave a positive reaction with 16 of 22 (73%) pneumococcal culture-positive spinal fluids. One false-positive result out of 45 pneumococcal culture-negative spinal fluids was also observed. D3114/63, an IgM MAb to non-PC determinants of teichoic acids, detected 15 of 22 of the pneumococcal culture-positive spinal fluids with one false-positive result. IgG2b and IgG3 anti-PC MAbs were less efficient than the IgM anti-PC MAb at detecting pneumococci in spinal fluids. Like the IgM anti-PC MAb, omniserum detected 73% of the culture-positive pneumococcal spinal fluids, with one false-positive result. The use of anti-PC or D3114/63 MAbs instead of a pooled serum such as omniserum has several advantages: (i) use of a single cross-reactive antibody rather than 83 pooled antibodies; (ii) possibility of a higher concentration of reactive antibody, which may increase the sensitivity of the test; (iii) a standardized antibody preparation; (iv) ease of preparation of the antibody; and (v) less expense.     

Role of outer membrane protein H (OmpH)- and OmpA-specific monoclonal antibodies from hybridoma tumors in protection of mice against Pasteurella multocida.

Two major outer membrane proteins of Pasteurella multocida, designated OmpH and OmpA, were characterized and shown to be related to the families of porin and heat-modifiable proteins, respectively. The backpack hybridoma tumor system in BALB/c mice was used to continuously deliver immunoglobulin G2b (IgG2b) monoclonal antibodies (MAbs) specific for OmpH (MAb MT1) and OmpA (MAb MT4.1). MAbs were detected in serum and peritoneal lavage samples of mice bearing hybridoma tumors by an enzyme-linked immunosorbent assay and an immunoblot assay. Highly significant protection was observed in mice bearing MT1 hybridoma tumors against both intraperitoneal and intranasal challenge infections with homologous nontoxigenic P. multocida strains possessing MAb MT1-reacting epitopes, whereas the mice bearing MT4.1 hybridoma tumors were not protected. The numbers of P. multocida organisms in the lungs of mice bearing MT1 hybridoma tumors were significantly less than those in lungs of mice bearing MT4.1 hybridoma tumors at 48 h postchallenge. These results indicate that the OmpH-specific MAb inhibited proliferation of P. multocida in the lungs. MAb MT1 was unable to kill P. multocida in vitro in the presence of complement. However, an enhanced phagocytosis by polymorphonuclear cells (PMNs) was observed in mice bearing MT1 hybridoma tumors. P. multocida induced a more extensive and rapid influx of PMNs into the peritoneal cavity of mice bearing MT1 hybridoma tumors than of mice bearing MT4.1 hybridoma tumors. The results of this study demonstrate for the first time that IgG MAbs against OmpH of P. multocida are involved in the protection of mice against lethal challenge infection by means of opsonization and inhibition of proliferation of P. multocida as a result of increased influx of PMNs into the infection site.     

Protection against Brucella melitensis or Brucella abortus in mice with immunoglobulin G (IgG), IgA, and IgM monoclonal antibodies specific for a common epitope shared by the Brucella A and M smooth lipopolysaccharides.

Mice passively immunized prior to a challenge infection with immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) specific for a common epitope of both A- and M-dominant strains had viable Brucella abortus 544 or Brucella melitensis H38 counts in the spleen reduced to the same extent as did mice passively immunized with MAbs specific for either the A or the M epitope. The IgA MAb was not effective.     

Dog immunoglobulins. II. The antibacterial properties of dog IgA, IgM and IgG antibodies to Vibrio cholerae.

Secretory IgA antibodies to Vibrio cholerae were purified from the parotid saliva and mammary secretions of locally and orally immunized dogs using gel filtration, ion-exchange chromatography and anti-immunoglobulin immuno-absorbents. IgM and IgG antibodies were isolated from serum by gel filtration and ion-exchange chromatography. IgA antibodies proved to have minimal, if any, activity in direct killing of bacteria in the presence of complement or in the promotion of phagocytosis. The minimal activity which IgA had in these assays could be accounted for by extremely small quantities of IgM antibody. The same IgA antibodies, mixed with the challenge innoculum of Vibrio cholerae and fed to infant mice, protected these mice as efficiently as IgG or IgM antibodies.     

Protective Immunity of Pneumococcal Glycoconjugates

Pneumococcal polysaccharides (PSs), designated as T-cell independent type 2 (TI-2) antigens, induce poor immune responses in young children. Splenic marginal zone B cells, associated with CD21, CD19 and C3d, play an important role in TI-2 antibody responses, and provide host defense against bacterial pathogens. Antibody response, avidity, and opsonophagocytic activity of antisera were examined in mice immunized with type 9V PS conjugated to inactivated pneulmolysin (Ply) or to autolysin (Aly). Compared to mice given 9V PS alone, serum IgG and IgM concentrations against the 9V PS were higher in mice immunized with conjugates. High concentrations of serum antibodies were maintained for over 12 weeks. The relative avidities of IgG and IgM antibodies and opsonophagocytic activity against 9V pneumococci were high in mice immunized with conjugates. Thus, conjugate vaccines can induce high as well as long duration of antibody response and effective functional activity. In another study, mice received intranasal immunization with type 9V conjugate or 9V PS. These animals produced 9V PS IgG and IgA antibodies in their serum, spleen, intestine, lung, Peyer's patch and fecal extract samples. Mice immunized with these glycoconjugates exhibited opsonophagocytic activity and rapid bacterial clearance from blood and provided homologous and cross-protection against challenge with virulent pneumococci. These results indicate that intranasal immunization with glycoconjugate vaccines may serve as an alternative and convenient approach for prevention of pneumococcal infection.     

Protective immunity of pneumococcal glycoconjugates

Pneumococcal polysaccharides (PSs), designated as T-cell independent type 2 (TI-2) antigens, induce poor immune responses in young children. Splenic marginal zone B cells, associated with CD21, CD19 and C3d, play an important role in TI-2 antibody responses, and provide host defense against bacterial pathogens. Antibody response, avidity, and opsonophagocytic activity of antisera were examined in mice immunized with type 9V PS conjugated to inactivated pneulmolysin (Ply) or to autolysin (Aly). Compared to mice given 9V PS alone, serum IgG and IgM concentrations against the 9V PS were higher in mice immunized with conjugates. High concentrations of serum antibodies were maintained for over 12 weeks. The relative avidities of IgG and IgM antibodies and opsonophagocytic activity against 9V pneumococci were high in mice immunized with conjugates. Thus, conjugate vaccines can induce high as well as long duration of antibody response and effective functional activity. In another study, mice received intranasal immunization with type 9V conjugate or 9V PS. These animals produced 9V PS IgG and IgA antibodies in their serum, spleen, intestine, lung, Peyer's patch and fecal extract samples. Mice immunized with these glycoconjugates exhibited opsonophagocytic activity and rapid bacterial clearance from blood and provided homologous and cross-protection against challenge with virulent pneumococci. These results indicate that intranasal immunization with glycoconjugate vaccines may serve as an alternative and convenient approach for prevention of pneumococcal infection.     

Modulation of the lung inflammatory response to serotype 8 pneumococcal infection by a human immunoglobulin m monoclonal antibody to serotype 8 capsular polysaccharide

The human monoclonal antibody to serotype 8 pneumococcal capsular polysaccharide D11 [immunoglobulin M(kappa)] protects wild-type and complement component 4 knockout (C4 KO) mice against lethal intratracheal challenge with serotype 8 pneumococcus, but it does not promote polymorphonuclear leukocyte (PMN)-mediated pneumococcal killing in vitro. In this study, we investigated the effect of D11 on the blood and lung bacterial burdens and the serum and lung expression of inflammatory chemokines and cytokines in an intratracheal challenge model with serotype 8 pneumococcus in C4 KO mice. Pneumococcus was not detected in the blood of D11-treated mice, whereas control mice had high-grade bacteremia with >10(7) CFU. Control mice had a >5-log increase in lung CFU and D11-treated mice manifested a nearly 3-log increase in lung CFU compared to the original inoculum 24 h after infection. Serum and lung levels of soluble macrophage inflammatory protein 2 (MIP-2) and interleulin-6 (IL-6) as measured by an enzyme-linked immunosorbent assay were lower in D11-treated mice than in control mice 24 h after infection. Real-time PCR was performed to examine lung mRNA chemokine and cytokine expression. The results showed that D11-treated mice had significantly less gamma interferon, MIP-2, IL-12, monocyte chemoattractant protein 1/JE, and tumor necrosis factor alpha expression than control mice 24 h after infection. Histopathology and immunohistochemical staining of lung tissues revealed that D11-treated mice had less inflammation, fewer PMNs, and less myeloperoxidase staining than control mice 24 h after infection. These findings suggest that the efficacy of certain serotype-specific antibodies against pneumococcal pneumonia could be associated with modulation of the lung inflammatory response and a reduction in host damage.     

Monoclonal IgM antibodies from cytomegalovirus-infected mice recognize the GlcNAc-containing receptor determinant of murine CMV as well as neutralizing anti-CMV IgG antibodies.

This study examines monoclonal antibodies derived from mice at different time points after infection with attenuated murine cytomegalovirus (MCMV). The antibodies obtained from mice at 3 weeks p.i. were of IgG type (designated V-antibodies) and several could neutralize the virus. Those obtained at 5 weeks p.i. were of the IgM class (designated R-antibodies), bound to uninfected (MEF, mouse embryo fibroblast) cells, and thereby blocked MCMV plaque formation. In ELISA, the IgM monoclonals (R-antibodies) bound to GalB1-3GlcNAc and GalB1-4GlcNAc, the receptor determinants for MCMV. Similarly, these GlcNAc-containing residues blocked the binding of purified IgM monoclonal antibodies (MAbs) to MEF. The R- and V-series of antibodies showed mutual binding activities; for example, IgM MAb R-2D8 bound specifically to four (V-8C4, V-1C7, V-8C7, V-9C5) of six neutralizing IgG MAbs in ELISA. The same neutralizing IgG MAbs bound to antireceptor IgM antibodies in an immunoblot assay. This suggests that the IgM monoclonals directed against the known cell surface receptor determinant are anti-idiotypic antibodies against neutralizing antiviral IgG antibodies. The neutralizing antiviral IgG MAbs bound to 60- and 66-kDa MCMV polypeptides on Western blots, suggesting that these viral polypeptides may be important in MCMV binding to this receptor. The R-series might represent anti-idiotype antibodies capable of down-regulating antiviral V-antibodies and may also represent a mechanism for the induction of IgM autoantibodies directed at cell surface glycolipids present in autoimmune CMV-associated neuropathies.     

Effect of immunoglobulin G isotype on the infectivity of Chlamydia trachomatis in a mouse model of intravaginal infection.

It has been previously shown with an in vitro neutralization system that monoclonal antibodies (MAbs) to the major outer membrane protein (MOMP) of Chlamydia trachomatis, depending on the isotype of the MAb and the host cell used, can either neutralize or enhance the infectivity of this organism. MAbs to variable domain 4 (VD 4) of MOMP have been described that neutralize the infectivity of C. trachomatis when tested in a system in which either the host cell does not have detectable Fc gammaRIII receptors or complement is added to block the interaction of the MAb with the receptor. However, if Fc gammaRIII receptors are available, immunoglobulin G2b (IgG2b) MAbs to the VD 4 are able to enhance the infectivity of this pathogen. Two MAbs that recognize the sequence TLNPTIA in VD 4 of the MOMP but differ in isotype, E4 (IgG2b) and E21 (IgG1), were used to test whether in vivo the isotype of the MAb modulates the outcome of a vaginal infection in a murine model. A third MAb, CP33 (IgG2b), that recognizes the chlamydial lipopolysaccharide but does not neutralize infectivity of C. trachomatis, was also tested. Elementary bodies (EBs) of C. trachomatis, serovar E (BOUR), were pretreated with the three MAbs and were used to inoculate the vaginas of C3H/HeJ mice which had been pretreated with progesterone. Subsequently mice were monitored over a 5-week period with vaginal cultures. In the groups that were inoculated with EBs pretreated with MAbs directed to VD 4 of MOMP, there was a significant decrease (P < 0.05) in the number of mice infected. Only 30% of the mice were infected in the MAb E4-treated group, and 10% were infected in the MAb E21 group. This was in contrast to the groups inoculated with EBs pretreated with MAb CP33 and control untreated EBs, which resulted in 100 and 79% of the mice infected, respectively. Therefore, in this setting in which EBs were introduced in vivo coated with MAb, there was no enhancement of infection by IgG2b MAbs; rather, the results paralled the in vitro neutralization results, in which cells lacking Fc gammaRIII receptors were employed. Mice were also given the MAbs, as well as purified IgG as a control, by intraperitoneal injection before and after intravaginal inoculation with C. trachomatis. Despite relatively high levels of MAbs in serum and detectable levels of MAbs in the vagina at the time of infection, there was only modest protection in animals receiving MAb E21, with 60% of the mice infected in contrast to 90% of the mice receiving MAb E4, MAb CP33, and IgG. However, by the second week of infection compared to controls, there was a significant increase (P < 0.05) in the amount of chlamydiae recovered from the vaginas of mice that had received the two IgG2b MAbs, E4 and CP33. In summary, the presence of IgG2b MAbs directed to surface components of C. trachomatis at certain times during the course of infection may play a role in enhancing the infectivity of this pathogen.     

Murine immune response to Neisseria meningitidis group C capsular polysaccharide: analysis of monoclonal antibodies generated in response to a thymus-independent antigen and a thymus-dependent toxoid conjugate vaccine

Antibody (Ab) responses to polysaccharides (PSs) such as Neisseria meningitidis group C PS (MCPS) are characterized as being thymus independent (TI) and are restricted with regard to clonotype and isotype expression. PS conjugated to proteins, e.g., MCPS coupled to tetanus toxoid (MCPS-TT), elicits a thymus-dependent (TD) response. In order to understand the influence of the form of a vaccine (TI versus TD) on the Ab repertoire, we generated monoclonal antibody (MAb) panels from mice immunized and boosted with MCPS or MCPS-TT in different ways. The panels of MAbs were examined for isotype, fine specificity, affinity, and V(H) gene family usage. The use of MCPS-TT resulted in a shift in the isotype from immunoglobulin M (IgM) and IgG3 elicited in response to the MCPS to primarily IgG1. This isotype shift was accompanied by a change in the fine specificity of the response to the conjugate compared to that of PS. New fine specificities and increased affinity were observed in response to the TD antigen (Ag). Dot blot and Northern analyses of MCPS MAbs revealed that V(H) gene family usage is dominated by V(H)J558, used by 23 of 39 MAbs. V(H)3609 was seen in three MAbs of restricted fine specificity. V(H)Q52, V(H)7183, and V(H)VGAM3-8 were seen in more than one MAb across these panels, while V(H)10 and V(H)X24 were detected only once in response to the TI-2 Ag. All MAbs in the panels utilized kappa light chains, and all functional J(kappa) genes were expressed.     

Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis.

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain A1. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous A1 LOS, 12 immunotype LOSs of N. meningitidis (L1 through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous A1 LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the A1 LOS and with several or many of the 12 LOSs. ELISA inhibition at 50% showed that the inhibitory activities of the LOSs from strains A1 and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (L8) LOS. When compared with MAb 2-1-L8 (L8) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains A1 and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (L8) LOS. The expression of the L8a epitope in the A1 LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain A1 and the L8 prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis.     

Specificity and mechanism of immunoglobulin M (IgM)- and IgG-dependent protective immunity to larval Strongyloides stercoralis in mice

Protective immunity in mice to the infective third-stage larvae (L3) of Strongyloides stercoralis was shown to be dependent on immunoglobulin M (IgM), complement activation, and granulocytes. The objectives of the present study were to determine whether IgG was also a protective antibody isotype and to define the specificity and the mechanism by which IgG functions. Purified IgG recovered from mice 3 weeks after a booster immunization with live L3 was shown to transfer high levels of protective immunity to na?ve mice. IgG transferred into mice treated to block complement activation or to eliminate granulocytes failed to kill the challenge larvae. Transfer of immune IgG into IL-5 knockout (KO) mice, which are deficient in eosinophils, resulted in larval attrition, while transfer into FcRgamma KO mice did not result in larval killing. These findings suggest that IgG from mice immunized with live L3 requires complement activation and neutrophils for killing of L3 through an antibody-dependent cellular cytotoxicity (ADCC) mechanism. This is in contrast to the results of investigations using IgM from mice immunized with live L3 and IgG from mice immunized with larval antigens soluble in deoxycholate in which protective immunity was shown to be ADCC independent. Western blot analyses with immune IgM and IgG identified few antigens recognized by all protective antibody isotypes. Results from immunoelectron microscopy demonstrated that the protective antibodies bound to different regions in the L3. It was therefore concluded that while IgM and IgG antibodies are both protective against larval S. stercoralis, they recognize different antigens and utilize different killing mechanisms.     

Monoclonal antibodies against Candida tropicalis mannan: antigen detection by enzyme immunoassay and immunofluorescence.

Three strains of mice were immunized with Candida tropicalis cell walls, and antibodies against mannan were detected by indirect enzyme immunoassay (EIA) in 3 of 9 BALB/c mice, 4 of 11 C57BL/6 mice, and 4 of 8 CFW mice. Responding mice produced immunoglobulin M (IgM), but IgG was not detected in their sera. Fusion of the high-responder BALB/c mouse with a plasmacytoma cell line resulted in 41 clones secreting antimannan monoclonal antibodies (MAbs). Four clones selected for propagation included one IgM and one IgG MAb that reacted with mannans of Candida albicans serotypes A and B and of C. tropicalis and two IgM MAbs specific for an epitope only in the mannans of C. albicans serotype A and C. tropicalis. One of the IgM MAbs, CB6, was an effective substitute for rabbit antibodies in the double-antibody sandwich EIA to detect antigenemia produced in rabbits infected with C. albicans A or C. tropicalis. It could function either as the peroxidase-conjugated indicator antibody or as the capture antibody. Two MAbs, CB6 (C. tropicalis and C. albicans A specific) and AC3 (C. tropicalis and C. albicans A and B specific), functioned in place of polyclonal antisera in the serotyping of C. albicans by immunofluorescence. There was 95.8% agreement in the results of serotyping using MAbs as reagents compared with rabbit antisera. Competitive inhibition in EIA between CB6 and monospecific antisera against C. albicans factors 1, 4, and 6 indicated that CB6 binds to an epitope which is probably factor 6. Serologic similarity between factor 4 and the binding site of MAb AC3 was also determined.     

A monoclonal antibody specific for immunoglobulin A receptor triggers polymorphonuclear neutrophil superoxide release.

An immunoglobulin M (IgM) monoclonal antibody, My43, specific for IgA Fc receptor (Fc alpha R) on human monocytes, bound to human polymorphonuclear neutrophils (PMNs) and inhibited their ability to bind IgA but not IgG. It was observed that the PMN oxidative burst was induced by both polymeric IgA and aggregated IgG, whereas IgM was without effect. The IgG-mediated oxidative burst was inhibited by anti-Fc gamma RII Fab and anti-Fc gamma RIII F(ab')2 but not by My43. Conversely, the IgA-mediated oxidative burst was inhibited by My43 but not by anti-Fc gamma RII or anti-Fc gamma RIII. When anti-Fc receptor monoclonal antibodies (mAbs) were used directly as ligands, it was observed that both anti-Fc gamma RII Fab and anti-Fc gamma RII F(ab')2 promoted the oxidative burst when cross-linked. Moreover, My43, when cross-linked with F(ab')2 antimouse IgM, also triggered the oxidative burst, whereas an IgM anti-CD15 mAb, PM81, did not stimulate function. This demonstrates that IgA receptors on PMNs are function-triggering molecules and that an anti-IgA receptor mAb may be substituted as a ligand.