Expression of Fc gamma RIII on HeLa 229 cells: possible effect on in vitro neutralization of Chlamydia trachomatis.

The neutralizing activities of a murine immunoglobulin G3 (IgG3) monoclonal antibody specific for the major outer membrane protein of Chlamydia trachomatis and its monovalent Fab fragments were studied by using Syrian hamster kidney (HaK) cells and human epithelial (HeLa 229) cells. The intact IgG3 antibody was neutralizing for HaK cells but was nonneutralizing for HeLa cells. In contrast, monovalent Fab antibody fragments neutralized chlamydial infectivity for both HaK and HeLa cells. Immunofluorescence analysis of HeLa 229 cells with a panel of monoclonal antibodies specific to human Fc gamma receptors revealed the expression of cell surface Fc gamma RIII. We propose that Fc gamma RIII may obscure the chlamydia-neutralizing activity of certain IgG isotypes by facilitating the Fc gamma R-mediated entry of chlamydiae into HeLa 229 cells. These findings may help explain the inconsistencies that are commonly observed in results when HeLa 229 cells are used in chlamydia neutralization assays.     

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.     

Protective role of magnesium in the neutralization by antibodies of Chlamydia trachomatis infectivity.

Neutralization of the infectivity of Chlamydia trachomatis was assessed by using polyclonal antisera and monoclonal antibodies (MAbs). Polyclonal antisera and a species-reactive MAb as well as a subspecies-specific MAb, both of which were directed toward the major outer membrane protein of C. trachomatis, reduced the number of chlamydial inclusion-forming units in an in vitro assay. Neutralization was dependent on the presence of complement. The species-specific MAb reacted with all 15 serovars by a microimmunofluorescence assay and a dot blot enzyme-linked immunosorbent assay with heat-treated elementary bodies. On the other hand, this same MAb reacted with all serovars, except those in the C complex, by the dot blot enzyme-linked immunosorbent assay with viable organisms and neutralized in vitro all 10 serovars tested, except those in the C complex. When neutralization assays were performed in a solution containing Mg2+, neutralization by both polyclonal antisera and MAbs was significantly reduced. A dose response to Mg2+ supplied as MgSO4 revealed that all concentrations tested from 50 to 800 microM had some effect. Concentrations of greater than or equal to 400 microM MgSO4 completely abolished neutralization at the lowest dilution of polyclonal antisera and species-reactive MAb tested. Although Mg2+ also blocked the neutralization effect of the subspecies-specific MAb, this neutralization was not as complete as that observed with the species-reactive MAb. Addition of Mg2+ to the assay over the initial 45 min of incubation of C. trachomatis with MAb and complement showed that the organisms could be rescued to some extent over the first 30 min of incubation, after which time neutralization of infectivity could not be reversed. C. trachomatis treated with Mg2+, the species-reactive MAb, and complement were lethal to mice in an in vivo toxicity and infectivity assay, whereas mice injected with organisms incubated with the same MAb and complement without Mg2+ survived.     

Expression of IgG Fc receptor antigens in placenta and on endothelial cells in humans. An immunohistochemical study.

Expression of leukocyte IgG Fc receptor (Fc gamma R) antigens by placenta, endothelial cells (EC) of normal tissues, and ECs of kidney and skin from subjects with immune complex diseases was studied immunohistochemically using anti-Fc gamma R monoclonal antibodies (MAb). Monoclonal antibodies against all three leukocyte Fc gamma R classes stained placental villous macrophages. Placental villous trophoblasts were stained intensely by anti-Fc gamma RIII MAb 3G8, while both anti-Fc gamma RI (MAb 32) and anti-Fc gamma RII MAbs IV3, KU79, CIKM5, 2E1, KB61, and 41H16) antibodies did not react with these cells. Anti-Fc gamma RII MAbs IV3, KU79, CIKM5, 2E1, KB61, and 41H16 immunostained placental villous capillary EC, in contrast to anti-Fc gamma RI MAb 32 and anti-Fc gamma RIII MAb 3G8, CLB-Granl, and B73.1, which did not bind. Anti-Fc gamma RI MAb 32, anti-Fc gamma RII MAb IV3 and CIKM5, and anti-Fc gamma RIII MAb 3G8 did not react with the ECs of tonsil, liver, kidney, spleen, intestine, lung, or uterus. Similarly no EC staining was seen with these four MAbs in 14 skin and 14 kidney biopsies from subjects with immune-complex diseases. Fc gamma R antigens are expressed constitutively only by placental villous ECs and are not induced on nonplacental ECs by immune-complex-mediated diseases.     

Neutralization of Chlamydia trachomatis: kinetics and stoichiometry.

Monoclonal antibodies to the major outer membrane protein of Chlamydia trachomatis were used to neutralize C. trachomatis infectivity in HeLa 229 cells and to determine the kinetics and stoichiometry of the reaction. In vitro neutralization of C. trachomatis infectivity proceeded as a first-order reaction and required an activation energy of approximately 20 kcal/mol (ca. 84 kJ/mol). The rate of neutralization was linear with respect to antibody concentration and reaction temperature. The efficiency of neutralization decreased exponentially as the ratio of noninfective to infective chlamydiae increased in the antigen preparation. The neutralization assay was also significantly affected by reaction parameters such as the reaction volume and the duration of incubation. Stoichiometric calculations showed that an average ratio of 10(3) and 10(4) immunoglobulin molecules per chlamydial particle was required to yield 50% neutralization by monoclonal antibodies specifying serovar-specific and species-specific epitopes, respectively. The implications of these findings for vaccine design and for the role of the major outer membrane protein in the pathogenesis of chlamydial infections are discussed.     

Mimicry of a neutralizing epitope of the major outer membrane protein of Chlamydia trachomatis by anti-idiotypic antibodies.

The major outer membrane protein (MOMP) is a primary target antigen for the development of chlamydial vaccine. This protein is composed of four variable domains (I to IV) flanked by constant regions. Some of the variable domains contain antigenic determinants that elicit a neutralizing antibody response. Murine monoclonal antibodies (MAbs) against three nonoverlapping epitopes of MOMP were developed. One of these, called DP10, bound to all serovars, as shown by immunoblot analysis, and neutralized chlamydial infectivity for hamster kidney (HaK) cells in a complement-independent in vitro assay. Furthermore, analysis of the fine specificity of this MAb showed that it recognized a synthetic peptide contained within variable domain IV of the MOMP. Anti-idiotypic antibodies (aId) directed against this anti-MOMP MAb were produced in rabbits. These aId specifically bound to the relevant idiotype (DP10) and inhibited the binding of anti-MOMP MAb (DP10) to MOMP preparations in a dose-dependent fashion. The specificity of our aId for the binding site of anti-MOMP MAb is further suggested by the binding inhibition of affinity-purified aId to DP10 by the synthetic peptide defined by the idiotype. In addition, these aId also reacted with anti-MOMP antisera from rats and mice, suggesting an idiotypic cross-reactivity between these species. Finally, immunization of naive mice with aId induced an antibody response directed against the peptide defined by our anti-MOMP MAb and with neutralizing activity. Taken together, these data suggest that aId mimic a neutralization site on MOMP and could serve as a surrogate antigen to induce protective immunity against Chlamydia trachomatis.     

Monoclonal antibody neutralization of unmanipulated Chlamydia trachomatis serovar A infection of human epithelioid cells (A-431)

A human epithelioid cell line (A-431) was tested in parallel with McCoy fibroblast cells for the growth of trachoma-related serovar A Chlamydia trachomatis without centrifugation or cycloheximide addition. A-431 cells were 4–7 times more susceptible to infection with serovar A than McCoy cells in such unmanipulated cultures. Murine monoclonal antibodies (MAbs) developed against serovar A were then evaluated for their ability to inhibit unmanipulated serovar A infectivity of A-431 cells. Two of seven MAbs tested neutralized infectivity by more than 50%. An IgG2a MAb (2C8) that is specific for serovar A, and another IgG2a MAb (4E3) that reacts equally with serovars A and L2 neutralized infectivity of serovar A by 72.2 ± 3.7% and 56.0 ± 5.8% (mean ± SEM of 7 experiments) respectively. Mouse immune serum (MIS) raised against serovar A elementary bodies (EB) neutralized infectivity of serovar A by 76.0 ± 4.9% (mean ± SEM of 7 experiments). Immunoblot detection of serovar A EB polypeptides separated by SDS-PAGE indicated that 2C8 reacted with a 16 kD and 4E3 reacted with a 12 kD polypeptide while MIS reacted with several polypeptides including the major outer membrane protein (MOMP). These studies show that the human epithelioid cell line A-431 is a more susceptible host than McCoy cells in unmanipulated cultures, and that 2 MAbs neutralize serovar A infectivity of A-431 cells. Identification of antigenic moieties of importance in unmanipulated chlamydial infections may help in the development of potential vaccines against trachoma.     

The synergistic neutralization of Rift Valley fever virus by monoclonal antibodies to the envelope glycoproteins

Summary A panel of monoclonal antibodies (MAbs) mapping to different antigenic sites on the RVFV G 1 and G 2 proteins were used to examine the mechanisms involved in neutralization of the virus. Three types of synergistic neutralization of RVFV were observed on mixing various pairs of MAbs. Firstly, enhanced neutralization occurred for two MAb pairs that showed augmented binding for G 2. These comprised a combination of a neutralizing MAb with a non-neutralizing antibody, as well as two antibodies which were non-neutralizing individually. In the second category, synergistic neutralization was observed between combinations of MAbs for which increased binding had not been detected. Lastly, mixtures of G 1 and G 2-specific MAbs were also capable of enhancing neutralization. Post-adsorption neutralization assays revealed that some MAbs neutralized cell-attached virus efficiently, indicating that they can neutralize by inhibiting the infection process after virus attachment. MAbs mapping to G 1 II e, G 2 I b and G 2 I c were unable to neutralize adsorbed virus and thus probably neutralize by preventing virus attachment to cells. Several G 1-reactive MAbs displayed low level post-adsorption activity, suggesting they may be capable of inhibiting RVFV infectivity at different stages of the replication cycle.     

In vitro mechanisms of monoclonal antibody neutralization of alphaviruses

We have previously identified at least eight epitopes on the E2 glycoprotein of Venezuelan equine encephalomyelitis (VEE) virus vaccine strain TC-83 by using monoclonal antibodies (MAbs). Several of these antibodies identified a critical neutralization (N) domain in competitive binding assays. Passive transfer of these MAbs protected animals from a lethal virus challenge. Using radioactive, purified virus as a marker, we have demonstrated that antibody-mediated virus N, preattachment, can be effected by one of three mechanisms. Interaction of antibody can block virus attachment to susceptible Vero or human embryonic lung cells. The MAbs that were most efficient at blocking attachment were those that defined epitopes spatially proximal to the E2c epitope. The E2c MAbs were, however, the most efficient antibodies for neutralizing virus postattachment. Other E2 MAbs were unable to efficiently block virus attachment to cells; however, resulting replication as monitored by plaque assay or intracellular viral RNA synthesis could not be detected. One novel MAb that defined the E2f epitope appeared to enhance virus attachment to Vero cells, but not BHK-21 or LLC-MK2 cells, by stabilizing virus-cell interaction. This antibody did, however, efficiently neutralize virus infectivity. Once virus had attached to cells, the ability of most MAbs to neutralize infectivity was diminished, except for E2c MAbs. On a molar basis antibody Fab fragments were less efficient than intact antibody at blocking virus attachment.     

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.     

A human CD4+ T-cell line expresses functional CD64 (Fc gamma RI), CD32 (Fc gamma RII), and CD16 (Fc gamma RIII) receptors but these do not enhance the infectivity of HIV-1-IgG complexes.

T cells do not generally express Fc receptors (FcRs). However, we report here that C8166 cells, a human CD4+ T lymphoblastoid cell line, widely used in research into the human immunodeficiency virus type 1 (HIV-1), expressed CD64 (Fc gamma RI), CD32 (Fc gamma RII), and CD16 (Fc gamma RIII) on the plasma membrane as shown by immunostaining with specific monoclonal antibody fragments. Another human CD4+ T lymphoblastoid cell line. H9, expressed none of these FcRs. C8166 cells bound monomeric normal rat serum IgG in a dose-dependent manner, and when saturated bound heat-complexed immunoglobulin G (IgG) also dose dependently. These observations are consistent with the presence on the C8166 T-cell line of both high- and low-affinity Fc gamma Rs. Fc gamma Rs are putative receptors for virus-IgG complexes, but in this study did not enhance infectivity of HIV-1 complexed with a human neutralizing mAb or three rat neutralizing mAbs. Virus complexed with a non-neutralizing mouse mAb was unable to infect cells using Fc gamma Rs as receptors after CD4 was blocked with a specific anti-CD4 mAb.     

Characterization of a neutralizing monoclonal antibody directed at variable domain I of the major outer membrane protein of Chlamydia trachomatis C-complex serovars.

A monoclonal antibody (MAb), C10, that neutralized in vitro the infectivity of serovars C, I, J, and L3 (members of the C and C-related complexes) of Chlamydia trachomatis was identified. Of the 15 major serovars and the mouse pneumonitis strain of C. trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae, which were used as nontreated and heat-treated (56 degrees C, 30 min) antigens in a dot blot assay, only serovars C, I, J, and L3 were recognized with both the native and treated antigens. Western blot (immunoblot) results showed that MAb C10 recognized the major outer membrane protein of these four serovars. Overlapping hexameric peptides corresponding to variable domains (VDs) I, II, III, and IV of the major outer membrane protein of C. trachomatis serovar C were synthesized, and peptide screening showed that MAb C10 mapped to the VD I amino acid sequence VAGLQNDPT. Results of an in vitro neutralization assay correlated with those of the indirect immunofluorescence assay, Western blot, and dot blot assay in that only serovars C, I, J, and L3 were neutralized by MAb C10. In vitro competitive neutralization experiments, using a peptide representing VD I of serovar C to compete with C. trachomatis serovar C for MAb C10 binding, revealed that both serological and neutralizing activities of MAb C10 were inhibited by the VD I peptide. In an in vivo toxicity/infectivity assay using serovar L3 pretreated with MAb C10, there was 100% survival of mice infected with a lethal dose at 48 h. In contrast, the control group, consisting of mice injected with the same dose of L3 pretreated with a MAb that does not recognize L3, had no survivors during a 48-h observation period. In summary, since the surface-exposed contiguous epitope recognized by MAb C10 binds neutralizing antibodies that are subspecies specific for the C and C-related complexes, it should be considered for inclusion in the development of a chlamydial vaccine.     

Novel monoclonal antibodies that bind to wild and fixed rabies virus strains

Ten monoclonal antibodies (MAbs) against rabies virus, including IgG3κ, IgG2aκ, IgMκ, and an IgG2bκ isotype, were produced and characterized using neutralization, ELISA, immunodot-blot, and immunofluorescence assays. MAb 8D11, which recognized rabies virus glycoprotein, was found to neutralize rabies virus in vitro. When submitted to an immunofluorescence assay, seven MAbs showed different reactivity against 35 Brazilian rabies virus isolates. Three MAbs (LIA 02, 3E6, and 9C7) only failed to recognize one or two virus isolates, whereas MAb 6H8 was found to be reactive against all virus isolates tested. MAbs were also evaluated for their immunoreactivity against fixed rabies virus strains present in human and veterinary commercial vaccines. MAbs LIA 02, 6H8, and 9C7 reacted against all vaccine strains, while the remaining MAbs recognized at least 76% of vaccine strains tested. This research provides a set of MAbs with potential application for improving existing or developing new diagnostic tests and immunoassays.     

Monoclonal antibodies to pertussis toxin: utilization as probes of toxin function

Six monoclonal antibodies (MAbs) to pertussis toxin (PT) have been generated and characterized. Five of these MAbs (3CX4, 3C4D, 6D11C, 6FX1, and X2X5) interact with determinants on the catalytic subunit (S1) of PT, and one (6DX3) is specific for subunit S4. The MAbs are divided into three groups based upon their ability to neutralize the effects of PT in a Chinese hamster ovary (CHO) cell assay. Three of the MAbs (3CX4, 3C4D and 6D11C) had high neutralization titers, one MAb (6FX1) displayed weak neutralizing activity, and two MAbs (X2X5 and 6DX3) had no neutralizing ability. The combination of one of the high titer MAbs (3CX4) with the low titer MAb (6FX1) resulted in a synergistic enhancement of neutralizing capability. F(ab')2 fragments prepared from MAb's 3CX4 and X2X5 displayed activities in the CHO-cell assay which were identical to the native MAb's. The ability of the MAbs to neutralize PT in the CHO-cell toxin neutralization assay correlated with their ability to inhibit the in vitro ADP-ribosylation of PT. A competition ELISA method demonstrated that this panel of MAbs recognizes at least four separate epitopes on the PT molecule. Biotin-conjugated MAbs were shown to be useful reagents to probe the interaction of pertussis toxin with fetuin.     

The functional activity of Fc gamma RII and Fc gamma RIII on subsets of human lymphocytes.

Subsets of human lymphocytes were isolated from peripheral blood using magnetic beads coated with anti-CD4, -CD8, -CD19 or -CD56 antibodies to yield T4, T8, B and natural killer (NK) cell suspensions with greater than 95% purity. The functional activity of Fc gamma receptor II (Fc gamma RII) and Fc gamma receptor III (Fc gamma RIII) on these subsets was assessed by measuring rosette formation with red cells sensitized with known levels of either rabbit IgG or human (monoclonal or polyclonal) IgG1 anti-D, IgG3 anti-D or IgG3 anti-c (E-IgG). Lysis of red cells by K cells (mediated by Fc gamma RIII) in antibody-dependent cell-mediated cytotoxicity (ADCC) assays was promoted by polyclonal and some monoclonal antibodies. Using these 'ADCC+' antibodies, minimum red cell sensitization levels required to promote rosette formation with NK cells were 2000 IgG1 or IgG3 molecules/red cell compared to 15,000 IgG1 or 4000 IgG3 molecules/red cell with 'ADCC-' monoclonal antibodies. The greater efficiency of ADCC+ antibodies is consistent with their previously reported ability to bind Fc gamma RIII via CH2 and CH3 domains whereas ADCC- antibodies bind only via CH3 domains. B cells formed rosettes only at high levels of sensitization: approximately 60,000 IgG1 or 20,000 IgG3 anti-D molecules/cell. These data reflect the low affinity of Fc gamma RII for monomeric human IgG. Although over 90% of NK cells bound anti-CD16, and 70% formed rosettes with red cells sensitized with rabbit IgG (30,000 molecules/cell), only 25% of NK cells formed rosettes with E-IgG3 at 100,000 IgG molecules/cell. Approximately 35% of B cells, 10% of T8 cells but no T4 cells formed rosettes with E-IgG (100,000 IgG3 molecules/cell). With T8, B and NK cells, IgG3 anti-D promoted greater rosette formation than IgG1 anti-D at comparable levels of sensitization. Presumably the longer hinge region of IgG3 enabled it to bridge the gap between negatively charged lymphocytes and red cells more efficiently than IgG1.     

Neutralization of Chlamydia trachomatis infectivity with antibodies to the major outer membrane protein.

Rabbit immunoglobulin G (IgG) antibodies raised against the major outer membrane protein of the Chlamydia trachomatis lymphogranuloma venereum strain 434 neutralized the infectivity of the parasite for HeLa 229 cells. The mechanism by which anti-major outer membrane protein IgG prevented C. trachomatis from establishing infection was studied by using intrinsically 14C-radiolabeled elementary bodies. Neutralized elementary bodies were filterable through a polycarbonate filter (pore diameter, 600 nm), demonstrating that reduction in infectivity was not due to the aggregation of elementary bodies by cross-linking IgG. Antibody-neutralized elementary bodies attached to and penetrated HeLa cells at rats nearly identical to those for infectious organisms exposed to nonneutralizing control IgG. These results suggest that antibody interferes with the infectious process of the parasite after its internalization. Anti-major outer membrane protein Fab fragments could not be substituted for neutralizing IgG antibodies. The requirement for intact IgG implies that cross-linking of antibodies to the major outer membrane protein on the surfaces of the organisms may be instrumental in neutralization.     

Reactivity of cloned, expressed human Fc gamma RIII isoforms with monoclonal antibodies which distinguish cell-type-specific and allelic forms of Fc gamma RIII.

We have isolated and expressed a cDNA encoding human NK cell Fc gamma RIII. The NK cell cDNA differs from the neutrophil Fc gamma RIII cDNA by a number of point mutations and encodes an additional 21 amino acids at its C-terminus. When transiently expressed neutrophil and NK cell Fc gamma RIII were digested with N-glycanase, deglycosylated neutrophil Fc gamma RIII had a more rapid electrophoretic mobility than NK cell Fc gamma RIII, as is observed for the human Fc gamma RIII isoforms on normal cells. The neutrophil and NK cell Fc gamma RIII isoforms apparently result from cell-type specific expression of different forms of Fc gamma RIII mRNA. A TaqI RFLP was also found for human Fc gamma RIII. Monoclonal antibodies which have been used to distinguish the neutrophil and NK cell Fc gamma RIII isoforms and the NA1 and NA2 alleles of human neutrophil Fc gamma RIII were employed to study the expression of two Fc gamma RIII cDNA clones derived from neutrophils and NK cells. Fc gamma RIII encoded by the neutrophil-derived cDNA reacts with the monoclonal antibody CLBgran11, while the NK-cell Fc gamma RIII cDNA expresses the Fc receptor which carries an antigenic determinant recognized by the antibody GRM1. Characterization of hybrid Fc gamma RIII constructed by interchange of restriction fragments between the neutrophil and NK cell cDNAs allowed localization of antigenic determinants recognized by the monoclonal antibodies.     

An Fc gamma RII-, Fc gamma RIII-specific monoclonal antibody (2.4G2) decreases acute Trypanosoma cruzi infection in mice.

In order to study the role of Fc gamma Rs in Trypanosoma cruzi infection in mice, the 2.4G2 monoclonal antibody (MAb), specific to the extracellular domains of Fc gamma RII and Fc gamma RIII, was injected intraperitoneally into mice. Flow cytometry studies of uninfected mice showed that 2.4G2 MAb bound to peritoneal and lymph node cells, respectively, on days 2 and 6 after injection. Repeating 2.4G2 injections every 3 to 4 days decreased the availability of Fc gamma Rs on peritoneal, lymph node, and spleen cells. Injections of 2.4G2 MAb into T. cruzi-infected mice, at days -1, 3, 7, 11, 16, 20, and 24 relative to infection, reduced mortality in comparison with that in infected animals injected with an unrelated MAb (50 versus 93.3% mortality; P < 0.01). Parasitemia in 2.4G2-treated mice was significantly (three times) lower than in control animals on days 21 and 24 postinfection (P < 0.05), before parasite-specific antibodies were detectable at significant levels. Immunoglobulin and T. cruzi-specific antibody levels were similar in all groups of mice. These results indicate that repeated injections of 2.4G2 MAb administered to acutely infected mice reduce the in vivo infection level, suggesting that Fc gamma Rs play a role in the early host invasion by T. cruzi parasites.     

Protective efficacy of major outer membrane protein-specific immunoglobulin A (IgA) and IgG monoclonal antibodies in a murine model of Chlamydia trachomatis genital tract infection.

The protective efficacy of immunoglobulin A (IgA) and IgG monoclonal antibodies (MAbs) specific for the major outer membrane protein of Chlamydia trachomatis MoPn was evaluated in a murine genital tract infection model. MAbs were delivered into serum and vaginal secretions of naive mice by using the backpack hybridoma tumor system, and protective efficacy was assessed over the first 8 days following challenge by quantitative determination of chlamydial recovery from cervicovaginal swabs, histopathological evaluation of genital tract tissue, and immunohistochemical detection of chlamydial inclusions. IgA and IgG significantly reduced the incidence of infection following vaginal challenge with 5 50% infectious doses, but such protection was overwhelmed by 10- and 100-fold higher challenge doses. Both MAbs also consistently reduced vaginal shedding from infected animals with all three challenge doses compared with the negative control MAb, although the magnitude of this effect was marginal. Blinded pathological evaluation of genital tract tissues at 8 days postinfection showed a significant reduction in the severity of the inflammatory infiltrate in oviduct tissue of infected IgA- and IgG-treated animals. Immunohistochemical detection of chlamydial inclusions revealed a marked reduction in the chlamydial burden of the oviduct epithelium; this finding is consistent with the reduced pathological changes observed in this tissue. These studies indicate that the presence of IgA or IgG MAbs specific to major outer membrane proteins has a marginal effect in preventing chlamydial colonization and shedding from the genital tract but has a more pronounced effect on ascending chlamydial infection and accompanying upper genital tract pathology.     

Protection of wild-type and severe combined immunodeficiency mice against an intranasal challenge by passive immunization with monoclonal antibodies to the Chlamydia trachomatis mouse pneumonitis major outer membrane protein

Monoclonal antibodies (MAbs) to the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) were characterized for their ability to neutralize the infectivity of this organism in vitro and in vivo. One of the MAbs (MoPn-23) recognizes a nonlinear epitope in the MOMP, MAb MoPn-40 binds to a linear epitope in the variable domain 1 (VD1), and MAb MoPn-32 recognizes the chlamydial lipopolysaccharide. MAb MoPn-23 neutralized 50% of the infectivity of Chlamydia, as measured in vitro by using HAK (FcγIII⁻) and HeLa-229 (FcγIII⁺) cells at a concentration 100 times lower than MAb MoPn-40. MAb MoPn-32 had no neutralizing ability. In comparison to the control normal mouse immunoglobulin G, passive immunization of BALB/c mice with MAb MoPn-23 resulted in a highly significant protection against an intranasal (i.n.) challenge as determined by the change in body weight, the weight of the lungs, and the yield of Chlamydia inclusion-forming units (IFU) from the lungs. Passive immunization with MAb MoPn-40 resulted in a lower degree of protection, and MAb MoPn-32 afforded no protection. MAb MoPn-23 was also tested for its ability to protect wild-type (WT) and severe combined immunodeficient (SCID) C.B-17 mice against an i.n. challenge. Protection based on total body weight, lung weight, and yield of Chlamydia IFU was as effective in SCID as in WT C.B-17 mice. In conclusion, antibodies to MOMP can protect mice against a chlamydial infection in the presence or absence of T and B cells.