The characterization of four monoclonal antibodies specific for mouse IL-5 and development of mouse and human IL-5 enzyme-linked immunosorbent

Four rat mAb directed against mouse IL-5 have been characterized by their ability to remove and neutralize mouse IL-5 activity in various bioassays. All four mAb absorbed IL-5 activity from solution. Although all were able to neutralize mouse IL-5 bioactivity, two were significantly more effective. These two were also able to neutralize the activity of mouse IL-5 delivered to B cells during a cognate-linked interaction with a Th cell clone. A two-site sandwich ELISA specific for mouse IL-5 was developed by using pairs of mAb. The mouse IL-5 ELISA is capable of detecting natural or mouse rIL-5 in supernatants, crude bacterial lysates, and high concentrations of mouse serum, and has a detection limit of less than 20 pg. Two of these antibodies cross-reacted with and neutralized human rIL-5, and one of these was used for development of an ELISA for human IL-5.     

Immunization with an anti-idiotypic antibody against the broadly lipopolysaccharide-reactive antibody WN1 222-5 induces Escherichia coli R3-core-type specific antibodies in rabbits

The mouse monoclonal antibody (mAb) WN1 222-5 recognizes a carbohydrate epitope in the inner core region of LPS that is shared by all strains of Escherichia coli and Salmonella enterica and is able to neutralize their endotoxic activity in vitro and in vivo. Immunization of mice with mAb WN1 222-5 yielded several anti-idiotypic mAbs one of which (mAb S81-19) competitively inhibited binding of mAb WN1 222-5 to E. coli and Salmonella LPS. After immunization of rabbits with mAb S81-19, the serological responses towards LPS were characterized at intervals over two years. Whereas the serological response against the anti-idiotype developed as expected, the anti-anti-idiotypic response against LPS developed slowly and antibodies appeared after 200?d that bound to E. coli LPS of the R3 core-type and neutralized its TNF-α inducing capacity for human peripheral mononuclear cells. We describe the generation of a novel anti-idiotypic antibody that can induce LPS core-reactive antibodies upon immunization in rabbits and show that it is possible, in principle, to obtain LPS neutralizing antibodies by anti-idiotypic immunization against the mAb WN1 222-5. The mimicked epitope likely shares common determinants with the WN1 222-5 epitope, yet differences with respect to either affinity or specificity do exist, as binding to smaller oligosaccharides of the inner core was not observed.     

Monoclonal antibodies to human tumor necrosis factor alpha: in vitro and in vivo application.

Three stable murine hybridoma cell lines, which secrete monoclonal antibodies (mAb) to human tumor necrosis factor alpha (TNF alpha), were established. None of the monoclonal antibodies cross-reacted with lymphotoxin, interleukin 2 (IL 2) or Interferon gamma (IFN gamma). The highly species-specific monoclonal antibody, designated as mAb 195, neutralizes the cytotoxic activity of human and chimpanzee TNF alpha. This antibody was further used during in vivo studies to neutralize human TNF alpha in a murine animal model. The mAb 114 is a weakly neutralizing antibody that binds to a different epitope of TNF alpha than mAb 195. mAb 114 shows a wide range of cross-reactivity with TNF alpha of the following species: dog, pig, cynomolgus, rhesus, baboon and chimpanzee. The mAb 199 binds to human TNF alpha, but does not neutralize the cytotoxic activity. The epitope recognized by this mAb is in close proximity to mAb 114. A reproducible, sensitive immunoassay for human TNF7 alpha has been developed using the antibodies mAb 199 and mAb 195. The test is performed within 6 hr and detects TNF7 alpha in serum samples, with a limit of detection of 5 to 10 pg/mL.     

Recombinant monoclonal antibodies for rabies post-exposure prophylaxis

Rabies virus is a prototypical neurotropic virus that causes one of the most dangerous zoonotic diseases in humans. Humanized or fully human monoclonal antibodies (mAb) that neutralize rabies virus would be the basis for powerful post-exposure prophylaxis of rabies in humans, having several significant benefits in comparison with human or equine rabies polyclonal immunoglobulins. The most advanced antibodies should broadly neutralize natural rabies virus isolates, bind with conserved antigenic determinants of the rabies virus glycoprotein, and show high neutralizing potency in assays in vivo. The antibodies should recognize nonoverlapping epitopes if they are used in combination. This review focuses on basic requirements for anti-rabies therapeutic antibodies. The urgency in the search for novel rabies post-exposure prophylaxis and methods of development of anti-rabies human mAb cocktail are discussed. The rabies virus structure and pathways of its penetration into the nervous system are also briefly described.     

In vitro efficacy of anti-HIV immunotoxins targeted by various antibodies to the envelope protein.

Six different anti-HIV envelope antibodies and one irrelevant control antibody were coupled to ricin A chain and tested for their efficacy in inhibiting HIV tissue culture infections. The anti-HIV antibodies consisted of five monoclonals, three of murine and two of human origin, and one polyclonal preparation prepared by affinity purifying pooled serum antibodies from HIV-infected humans on rgp160. The binding specificity of the antibodies was defined by ELISA by using recombinant envelope proteins and synthetic peptides, and by flow cytometry on HIV-infected cells. The in vitro efficacy of the antibodies was tested by the abilities of the immunotoxins to inhibit protein synthesis in persistently infected cell lines and by their abilities to inhibit HIV production during both acute and persistent infection as measured with an HIV-specific focal immunoassay. The immunotoxins were tested against a panel of distinctly different HIV isolates. The results indicate the following: 1) A mAb to the immunodominant neutralizing loop was highly effective against homologous strains of HIV, but had no activity against heterologous HIV. 2) The efficacy of anti-gp41 mAb varied depending upon the epitope recognized and possibly the affinity of binding to gp41. 3) The polyclonal human anti-gp160 antibodies produced the immunotoxin with the broadest specificity for different HIV strains and the greatest specific activity. This is related to the polyclonal nature of the preparation rather than an increase in relative avidity of the antibody. 4) Activity of an immunotoxin is not a direct function of the binding of the antibody to the surface of infected cells. 5) The ability of an immunotoxin to halt the spread of infection through a tissue culture cell population is dependent upon the ability of the antibody to neutralize the virus as well as the activity of the toxin. Our data suggest that efficacious immunotoxins for the treatment of AIDS may be made with polyclonal anti-envelope antibodies derived from the serum of patients who have been infected with HIV or with appropriately chosen anti-gp41 antibodies.     

Anti-idiotype antibodies prevent antibody binding to mouse sperm and antibody-mediated inhibition of fertilization

Antibodies to components of sperm can interfere with sperm function and prevent fertilization by blocking specific steps of gamete interaction. It can be proposed that anti-idiotype antibodies (anti-Ids) that recognize determinants located close to or within the antigen-binding site of an anti-sperm antibody could block antibody binding to sperm antigen and antibody-mediated inhibition of fertilization. To test this hypothesis, rabbit polyclonal antibodies to idiotypic determinants of the monoclonal anti-sperm antibody M42.15 were developed and characterized. Previous studies demonstrated that M42.15 monoclonal antibody (mAb) inhibits fertilization in vitro and in vivo by inhibiting sperm-zona pellucida interaction. Anti-idiotype antibodies to M42.15 mAb (anti-Id M42) were isolated by affinity chromatography on immobilized M42.15 mAb. Binding specificity of anti-Id M42.15 was demonstrated in a solid-phase radioimmune binding assay and by specific immunoprecipitation of soluble M42.15 mAb. Anti-Id M42 competitively inhibited M42.15 mAb, but not P220.2 mAb, binding to mouse sperm, confirming that the anti-Id preparation contained antibodies directed against idiotopes within or adjacent to the antigen-binding site of the mAb. At equimolar concentrations, anti-Id M42 inhibited binding of 125I-labeled M42.15 mAb to sperm by greater than 80%. These results showed that anti-Id M42 efficiently blocked antibody binding to sperm and suggested that anti-Id M42 could be used to neutralize the anti-fertility activity of the M42.15 mAb. When tested in in vitro fertilization assays, anti-Id M42, but not rabbit immunoglobulin, prevented M42.15 mAb-induced inhibition of fertilization. Together, these results show that the inhibitory activity of anti-sperm antibodies capable of interfering with gamete interaction can be neutralized by anti-Id that recognize determinants close to the antigen-combining site of the anti-sperm antibody.     

Preparation and characterization of beta 1-bungarotoxin bispecific monoclonal antibody.

A hybrid hybridoma (tetradoma) that produces bispecific monoclonal antibodies (mAbs)2, which recognize two different epitopes on the A chain of beta 1-bungarotoxin (beta 1-bgt) at peptide sequences 46-51 and 100-106, has been obtained by fusing two hybridoma cell lines. The bispecific mAb were observed to inhibit 98% of the enzymatic activity of beta 1-bgt and neutralize its lethal toxicity completely. The avidity between the bispecific mAb and beta 1-bgt was noted to be 4.5 x 10(10) (liter/nmol), which is about 45-150 folds higher than the avidity of its two parental mAbs. All the soluble complexes, obtained from bispecific mAb and beta 1-bgt with different molar ratios, emerged in the void volume of size exclusion chromatography column, indicating multiple complexes of beta 1-bgt and bispecific mAb were formed. Based on these results, it indicated that the binding of bispecific mAb with its two epitopes on beta 1-bgt, which facilitates the immuno-complex formation and enhances the avidity, also highly neutralizes the biological activity of beta 1-bgt.     

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity

Background

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity.

Methods and Principal Findings

Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity.

Conclusions

The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.     

Structural analysis of human and macaque mAbs 2909 and 2.5B: implications for the configuration of the quaternary neutralizing epitope of HIV-1 gp120

The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 ? in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs.     

Treatment with GM-CSF and IL-2 in patients with metastatic colorectal carcinoma induced high serum levels of neopterin and sIL-2R,an indicator of immune suppression

Cytokines may enhance the effect of therapeutic monoclonal antibodies (mAb). Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) have been shown to increase ADCC levels. GM-CSF may augment the induction of an idiotypic network response (anti-tumour immunity). The clinical anti-tumour effect of a combination of mouse mAb17-1A-1A [anti-colorectal carcinoma (CRC)], and GM-CSF was, however, not enhanced by the addition of IL-2. In the present study, some immune functions considered to be involved in mAb-mediated tumour cell killing were analysed in patients receiving GM-CSF and GM-CSF/IL-2 respectively together with the mAb17-1A-1A. Ten patients received mAb17-1A and GM-CSF, and ten patients mAb17-1A with GM-CSF and IL-2. During a 10- day cytokine treatment period, a significantly higher increase in white blood cell counts was noted in the GM-CSF/IL-2 treatment group as compared to GM-CSF-treated patients. In the GM-CSF/IL-2 group, significantly higher serum concentrations of neopterin and soluble IL-2 receptor (sIL-2R) respectively were induced as compared to GM-CSF-treated patients. However, the ADCC of peripheral blood mononuclear cells (PBMC) against a CRC cell line was significantly higher in the GM-CSF group than in the GM-CSF/IL-2 group. The frequencies of patients developing human anti-mouse antibodies (HAMA) and anti-idiotypic antibodies were the same in both groups, while serum concentrations were significantly lower in the GM-CSF/IL-2 group as compared to the GM-CSF group. GM-CSF/IL-2 therapy seems to induce an immune suppressive stage compared to GM-CSF alone affecting cytotoxic mononuclear cells and B cells, which might be mediated through the neopterin metabolic pathway or other inducible immune suppressive factors such as reactive oxygen and nitrogen intermediates.     

Characterization of neutralizing mouse‐human chimeric and shuffling antibodies against botulinum neurotoxin A

Mouse‐human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2–4, 2–5, 9–4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V_H) and light chains (V_L) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO‐DG44 cells were transfected with these plasmids and mouse‐human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V_H or V_L domains were constructed. A shuffling antibody (AC2494) that derived its V_H and V_L domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high‐potency neutralizing chimeric antibodies by selecting and shuffling V_H and V_L domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.     

Development and characterization of monoclonal antibodies to murine macrophage colony-stimulating factor

The macrophage-specific CSF (CSF-1), purified from murine L cell-conditioned medium, supports the in vitro proliferation and survival of various murine mononuclear phagocyte colony-forming cells. In this report we describe the production and functional characterization of two monoclonal antibodies (mAb) to CSF-1 obtained from rat X rat hybridomas. These two mAb are functionally distinct and recognize different epitopes on CSF-1. The mAb 5A1 binds to and inhibits the biologic function of CSF-1, and the second mAb (D24) binds CSF-1 but does not neutralize its biologic activity. The mAb 5A1 inhibits colony formation of tissue mononuclear phagocyte colony-forming cells as well as the committed bone marrow stem cells for both granulocytes and monocytes. The extent of colony inhibition by mAb 5A1 is dependent on the tissue origin of colony-forming cells. CSF-1 complexed with mAb 5A1 does not bind to its cell surface receptor of peritoneal exudate macrophages, and mAb 5A1 does not complex with cell-bound CSF-1. Although both bone marrow cell-derived macrophages and J774.1 macrophages bind CSF-1, mAb 5A1 inhibits the proliferation of only bone marrow cell-derived macrophages. The non-neutralizing mAb D24 does not block binding of CSF-1 to its cellular receptor, and it recognizes cell-bound CSF-1.     

Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV-1-Infected Individuals

Background

The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine.

Methods and Findings

We immortalized IgG⁺ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity.

Conclusions

This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.     

Generation and characterization of neutralizing human recombinant antibodies against antigenic site II of rabies virus glycoprotein

The currently recommended treatment for individuals exposed to rabies virus (RV) is post-exposure prophylaxis (PEP) through the combined administration of rabies vaccine and rabies immune globulin (RIG). Human monoclonal antibodies (mAbs) that neutralize RV offer an opportunity to replace RIG for rabies PEP. Here, a combinatorial human Fab library was constructed using antibody genes derived from the blood of RV-vaccinated donors. Selections of this library against purified RV virions resulted in the identification of 11 unique Fab antibodies specific for RV glycoprotein. Of the Fab antibodies, five were converted to full human IgG1 format. The human IgG antibodies revealed high binding affinity and neutralizing activities against RV fixed strains through a rapid fluorescent focus inhibition test in vitro as well as the early stage protective function after exposure to RV infection in vivo. Furthermore, epitope mapping and binding competition analysis showed that all of obtained human neutralizing and protective antibodies were directed to the antigenic site II of RV glycoprotein. Our results provide not only important insight into the protective immune response to RV in humans, but also more candidates eligible for use in a mAb cocktail aimed at replacing RIG for rabies post-exposure prophylaxis.     

Structure-activity relationship study of human interleukin-3: role of the C-terminal region for biological activity.

A structure-activity relationship study of human interleukin-3 (huIL-3) was performed by functional analysis of huIL-3 deletion and substitution variants combined with epitope mapping of huIL-3 specific neutralizing monoclonal antibodies (mAb). Analysis of the huIL-3 variants was accomplished by defining their capacity to compete with wild-type huIL-3 for binding to the huIL-3 receptor and to induce the proliferation of the huIL-3 dependent cell line M-O7. HuIL-3 variants with either 14 amino acids (aa) deleted from the N-terminus or eight aa from the C-terminus retained full biological activity in vitro. An huIL-3 variant, with 18 N-terminal aa deleted, exhibited a greater than 7-fold reduced receptor binding capacity and proliferative activity. No biological activity could be detected with a variant where 22 C-terminal aa have been deleted. Neutralizing mAb recognizing presumed discontinuous epitopes failed to interact with the latter deletion variant indicating a possible location of their epitopes within the C-terminal region. Computer-aided structure prediction and sequence homology analysis of this region indicated the presence of an amphiphilic alpha-helix with highly conserved residues like Lys110 and Leu111. Substitution of Lys110 with either Glu or Ala resulted in variants with a 10-fold reduced activity in the receptor binding assay and the proliferation assay. Further variants, where Leu111 was substituted by Pro or Met, were totally inactive in these assays. Analysis of the binding of the two neutralizing mAb to these substitution variants showed that they did not bind to either of the Leu111 variants suggesting that Leu111 is part of an active site. Based on our results, a possible model for the structure of the huIL-3 molecule can be constructed with two active sites in close proximity.     

Monoclonal antibody approach to the relationship between immunological structure and biological activity of thyrotropin

In order to locate the domains involved in the biological activity of TSH and to get some insight in the relationship between immunological and biological properties of TSH, 24 monoclonal antibodies (mAb) to 11 different antigenic regions of hTSH were tested for both binding to hTSH and inhibition of hTSH stimulation of adenylate cyclase in human thyroid membranes. These mAb were also investigated for binding to bovine TSH (bTSH), and interference with bTSH binding to the receptor and stimulation of adenylate cyclase. Radioiodinated human TSH (hTSH) was incubated with increasing concentrations of mAb. Maximum hTSH binding by the various mAb ranged from 15-75% and was not related to the apparent affinity of the mAb for hTSH. Maximum inhibition by the mAb of hTSH stimulation of adenylate cyclase ranged from 3-92%. As compared to the antigenic map of hTSH, it was observed that mAb reacting with the same antigenic regions might display varying inhibition of hTSH. Nevertheless, it was clearly shown that the most potent inhibitors of hTSH stimulatory activity interacted with epitopes located on the alpha- and beta-subunits or expressed only by holo hTSH. Only 11 of the 24 mAb cross-reacted significantly with bTSH. Seven exhibited the same inhibition of hTSH and bTSH stimulatory activity; the four remaining mAb rather than to inhibit adenylate cyclase stimulation as observed with hTSH, did not interfere or even increased adenylate cyclase stimulation by bTSH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of the anti-gp120 antibody response during seroconversion to human immunodeficiency virus type 1.

We have studied the development of the antibody response to the surface glycoprotein gp120 of human immunodeficiency virus type 1 in three individuals who presented with primary human immunodeficiency virus type 1 infection syndrome. Serum anti-gp120 antibodies were first detected 4 to 23 days after presentation, after p24 antigen and infectious-virus titers in the peripheral blood had declined manyfold from their highest values. Whether anti-gp120 antibodies present at undetectable levels are involved in clearance of viremia remains unresolved. Among the earliest detectable anti-gp120 antibodies were those to conformationally sensitive epitopes; these antibodies were able to block the binding of gp120 monomers to soluble CD4 or to a human monoclonal antibody to a discontinuous epitope overlapping the CD4-binding site. Some of these antibodies were type specific to a degree, in that they were more effective at blocking ligand binding to autologous gp120 than to heterologous gp120. However, the appearance of these antibodies did not correlate with that of antibodies able to neutralize the autologous virus in vitro by a peripheral blood mononuclear cell-based assay. Antibodies to the V3 loop were detected at about the same time as, or slightly later than, those to the CD4-binding site. There was a weak correlation between the presence of antibodies to the V3 loop and autologous virus-neutralizing activity in two of three individuals studied. However, serum from the third individual contained V3 antibodies but lacked the ability to neutralize the autologous virus in vitro, even immediately after seroconversion. Thus, no simple, universal correlate of autologous virus-neutralizing activity in a peripheral blood mononuclear cell-based assay is apparent from in vitro assays that rely on detecting antibody interactions with monomeric gp120 or fragments thereof.     

Anti-CD4 and anti-CD2 monoclonal antibody infusions in subjects with multiple sclerosis. Immunosuppressive effects and human anti-mouse responses

The immunologic responses to anti-T4 and anti-T11 mAb infusions were assessed in subjects with chronic progressive multiple sclerosis as part of phase I clinical studies. Eight subjects received five daily infusions (0.2 mg/kg/day) of either anti-T11 (CD2) or anti-T4 (CD4) murine mAb. It was found that in vivo anti-T cell mAb infusions specifically suppress in vitro measurements of the human immune response; anti-T11 mAb blocked T cell activation via the CD2 SRBC-binding protein, and anti-T4 mAb infusions abolished PWM-induced Ig synthesis without lysis of the CD4+ T cell subpopulations. With repeated infusions of the anti-T11 mAb in three subjects, anti-mouse antibodies were found in the circulation. Although the majority of human anti-mouse antibody was not isotype specific, significant anti-idiotypic-like activity was observed after repeated infusions in two of three subjects. The human anti-mouse antibodies were almost exclusively of the IgG isotype. The magnitude of the human anti-mouse response was significantly less after administration of either anti-T11 or anti-T4 as compared with anti-T12 mAb. Murine anti-T cell mAb can provide a specific, benign form of acute immunosuppression in humans. However, repeated administration of these reagents in more chronic diseases can result in anti-Id-like antibodies that block binding of the anti-T cell mAb to the T cell surface.     

Context matters: The importance of dimerization-induced conformation of the LukGH leukocidin of Staphylococcus aureus for the generation of neutralizing antibodies

LukGH (LukAB) is a potent leukocidin of Staphylococcus aureus that lyses human phagocytic cells and is thought to contribute to immune evasion. Unlike the other bi-component leukocidins of S. aureus, LukGH forms a heterodimer before binding to its receptor, CD11b expressed on professional phagocytic cells, and displays significant sequence variation. We employed a high diversity human IgG1 library presented on yeast cells to discover monoclonal antibodies (mAbs) neutralizing the cytolytic activity of LukGH. Recombinant LukG and LukH monomers or a LukGH dimer were used as capture antigens in the library selections. We found that mAbs identified with LukG or LukH as bait had no or very low toxin neutralization potency. In contrast, LukGH dimer-selected antibodies proved to be highly potent, and several mAbs were able to neutralize even the most divergent LukGH variants. Based on biolayer interferometry and mesoscale discovery, the high affinity antibody binding site on the LukGH complex was absent on the individual monomers, suggesting that it was generated upon formation of the LukG-LukH dimer. X-ray crystallography analysis of the complex between the LukGH dimer and the antigen-binding fragment of a very potent mAb (PDB code 5K59) indicated that the epitope is located in the predicted cell binding region (rim domain) of LukGH. The corresponding IgG inhibited the binding of LukGH dimer to target cells. Our data suggest that knowledge of the native conformation of target molecules is essential to generate high affinity and functional mAbs.     

Inhibition of HIV infection by a novel CD4 domain 2-specific monoclonal antibody. Dissecting the basis for its inhibitory effect on HIV-induced cell fusion.

HIV use the CD4 molecule as their primary cellular receptor. Residues in the N-terminal domain (D1) of CD4 are crucial to HIV attachment through the gp120 envelope component. However, other regions of CD4 appear to be required subsequently for virus- and cell-cell fusion. Little is understood of the post-binding steps which may differ between HIV variants. We report a novel anti-CD4 mAb that does not block CD4/gp120 binding, but that does efficiently block both viral infection and cell-cell syncytia formation, and define its contact site as residues in CD4 D2 using both mouse/human CD4 chimeras and CD4 substitution mutants. We also investigated the basis for its antiviral effect. Using the CD4 D2 specific mAb, we identify another conserved step in HIV infection, as evidenced by its ability to neutralize a broad range of primary isolates and T cell-line passaged strains. Monovalent forms of the mAb were used to determine if its activity was due to masking of the D2 epitope, to steric inhibition, or bivalency. Our data indicate that both binding site and bivalency of the mAb underlie its potency. The need for bivalency is not simply explained by affinity, because monovalent forms can displace the intact mAb and reverse its protective effect. These results provide evidence that binding of the D2-specific mAb prevents structural alterations necessary for membrane fusion.