anti-HA mAb Research Articles

Fusion of influenza virus with the endosomal membrane is inhibited by monoclonal antibodies to defined epitopes on the hemagglutinin

Epitopes on the hemagglutinin (HA) of A/seal/Massachusetts/1/80 (H7N7) influenza virus were mapped by genetic analysis of variants selected with monoclonal antibodies (MAbs). Electron microscopic studies demonstrated that the sites and the directions to which hemagglutination-inhibiting (HI) MAbs and non-HI MAbs bound were different on the HA molecule. Morphological analysis revealed that HI MAbs blocked attachment of the virus to the cells, while non-HI MAbs did not. Virus particles bound with non-HI MAbs were then found in the intracellular vacuoles. Together with the electron microscopic findings, a fluorescence dequenching assay indicated that non-HI MAbs inhibited the fusion of virus with the intracellular vacuolar membrane. It was thus shown that non-HI neutralizing MAbs did not inhibit attachment of the virus to the host cell receptor, but inhibited the fusion step in intracellular vacuoles. The results support the hypothesis that anti-HA MAbs which lack HI activity neutralize viral infectivity by interfering with the low pH-induced conformational change in the HA molecule, resulting in inhibition of the fusion step in the viral replication process (Kida, H., Yoden, S., Kuwabara, M., Yanagawa, R., 1985. Interference with a conformational change in the HA molecule of influenza virus by antibodies as a possible neutralization mechanism. Vaccine 3, 219–222).

Activation of the PI3K effector protein kinase B following ligation of CD28 or Fas.

Engagement of CD28 is required for optimal T cell activation initiated through the TCRlCD3 complex and CD28 can regulate multiple cytokinelchemokine genes and promote cell survival [ 1, 21. In contrast, a member of the TNF receptor family termed Fas, participates in T cell activation-induced cell death (apoptosis) upon ligation by its natural ligand or by antibody [3]. I t has previously been demonstrated that CD28 binds to the prototypical PTWm homology 2 domain coupled member of the phosphatidylinositol 3-kinase (PI3K) family [2], leading to a major increase in P13K activity which is required for CD28-dependent IL-2 production in normal T lymphocytes [1,2]. In addition, we have also demonstrated that Fas stimulates PI3K via an Ick-dependent mechanism, although PI3K appears not to be required for Fasinduced apoptosis [4]. The downstream effector targets of PI3K remain poorly defined, although i t now appears that the PKC related lunase Akdprotein kinase B (PKB) is a major target of PI3K [5] and both PI3K and PKB have been demonstrated to be involved in the regulation of cell survival events [5]. Given the opposite functional outcomes of ligation of CD28 or Fas and their coupling to PI3K, we investigated the effects of CD28 and Fas on PKB activity. Jurkat cells were transfected with the plasmid DNA for HA-tagged PKB alone or with a constitutively active PI3K that was generated by membrane localisation of the p l lO subunit which is known to result in constitutive activation of the kinase [6]. The constitutively active PI3K construct (rCD2pllO) consists of a chimeric molecule in which the cytoplasmic domain of rat CD2 (rCD2) cell surface antigen has been replaced by the p l lO catalytic subunit of PI3K. A kinase dead version of this construct (rCD2pllORlP) contains an inactivating arginine to proline mutation at position 1130 and was used as a control [6]. Jurkat cells (1.5 x 107 cellsl0.5 ml) were transfected via electroporation and after 16 hours at 37C, transfected Jurkat cells were assessed for viability by trypan blue exclusion. Viable transfected Jurkat cells were washed, resuspended and aliquoted at 1 x 106 cellsl0.5 ml and stimulated with anti-CD28 mAb 9.3, Fas mAb C H l l or gently co-sedimented with 0.5 x lo6 CHO-B7.1+ cells in a volume of 1 ml by centrifugation at 200g for 10 seconds. Cell lysates were immunoprecipitated using the 12CA5 anti-HA mAb. Alternatively, untransfected Jurkat or freshly isolated normal T lymphocytes were stimulated as above and endogenous PKB immunoprecipitated using an anti-PKB polyclonal antibody. The immunoprecipitates were assayed for PKB activity as assessed by the in vitro phosphorylation of histone H2B. Samples were electrophoresed through acrylamide gels by SDS-PAGE. After electrophoresis, the upper part of the gel was transferred by electroblotting onto PVDF membranes and immunoblotted with anti-PKB Ab to confirm transfection andlor immunoprecipitation efficiency. The lower part of the gel was dried and phosphorylated histone H2B detected by autoradiography at -7ooc. T o demonstrate the expression of CD28 or rCD2 on transiently transfected cells and the effectiveness of both the transfection and the PKB asssay, certain Jurkat cells were transfected with plasmid DNA for HA-PKB and the PI3K c h i m e r i c c o n s t r u c t s C D 2 p l l O or rCD2pllOWP. Immunofluorescent staining and flow cytometry of the viable Jurkat cells 16 hours after transfection, using anti-CD28 mAb or mouse IgG for control staining followed by FITC-conjugated rabbit anti-mouse IgG, confirmed expression of CD28 on Jurkat cells transfected with either HA-PKB alone or with rCD2pl lOor

The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors.

The tissue-restricted GATA-4 transcription factor and Nkx2-5 homeodomain protein are two early markers of precardiac cells. Both are essential for heart formation, but neither can initiate cardiogenesis. Overexpression of GATA-4 or Nkx2-5 enhances cardiac development in committed precursors, suggesting each interacts with a cardiac cofactor. We tested whether GATA-4 and Nkx2-5 are cofactors for each other by using transcription and binding assays with the cardiac atrial natriuretic factor (ANF) promoter_the only known target for Nkx2-5. Co-expression of GATA-4 and Nkx2-5 resulted in synergistic activation of the ANF promoter in heterologous cells. The synergy involves physical Nkx2-5-GATA-4 interaction, seen in vitro and in vivo, which maps to the C-terminal zinc finger of GATA-4 and a C-terminus extension; similarly, a C-terminally extended homeodomain of Nkx2-5 is required for GATA-4 binding. Structure/function studies suggest that binding of GATA-4 to the C-terminus autorepressive domain of Nkx2-5 may induce a conformational change that unmasks Nkx2-5 activation domains. GATA-6 cannot substitute for GATA-4 for interaction with Nkx2-5. This interaction may impart functional specificity to GATA factors and provide cooperative crosstalk between two pathways critical for early cardiogenesis. Given the co-expression of GATA proteins and NK2 class members in other tissues, the GATA/Nkx partnership may represent a paradigm for transcription factor interaction during organogenesis.

Characterization of monoclonal antibodies to the hemagglutinin-esterase glycoprotein of a bovine coronavirus associated with winter dysentery and cross-reactivity to field isolates.

Seven hybridoma cell lines producing monoclonal antibodies (MAbs) to the hemagglutinin-esterase (HE) glycoprotein of bovine coronavirus (BCV) were obtained from BALB/c mice that were immunized with an enriched peplomeric fraction of the winter dysentery (WD)-associated strain BCQ.2590. The specificities of these MAbs to either the dimeric (140-kDa) or the monomeric (65-kDa) form of the HE glycoprotein were determined by Western immunoblotting experiments with purified virus and immunoprecipitation tests with [35S]methionine-labelled infected cell extracts. Four of these anti-HE MAbs inhibited the hemagglutinating activity of the virus and three weakly neutralized its infectivity in vitro. In addition, competition binding assays allowed for the definition of two independent antigenic domains (domains A and D) and two overlapping antigenic domains (domains B and C) for the HE glycoprotein of the WD-associated strain; epitopes located within antigenic domain A were not associated with hemagglutination inhibition (HAI) and virus neutralization activities. In HAI tests, the four anti-HA MAbs defined two distinct antigenic subgroups among 24 BCV field isolates that have been associated with either typical outbreaks of WD or neonatal calf diarrhea (NCD) in Quebec dairy herds from 1986 to 1996. The Quebec WD-associated strains of BCV, as well as some of the NCD-associated strains isolated since 1991, fell within a subgroup distinct from that of the prototype Mebus strain.

Human monocytes are unable to bind to or phagocytize IgA and IgG immune complexes formed with influenza virus in vitro.

An in vitro model system was used to investigate how human monocytes recognize influenza A/WSN(H1N1)-infected epithelial cells, and the role that anti-influenza IgA and IgG Abs play in this interaction. Pretreatment of the monocytes with neuraminidase or F(ab')2 fragments of a mAb against the Ca1 epitope on the hemagglutinin (HA) molecule inhibited monocyte adherence to the infected cells. This suggested that monocytes bound to the sialic acid binding site on the HA molecule and not to other viral or epithelial cell Ags. In the presence of neutralizing concentrations of intact Abs (human serum IgA, secretory IgA, IgG, or mouse anti-HA mAb), monocytes were unable to bind to influenza-infected epithelium or to phagocytize the IgA or IgG immune complexes formed with influenza virus, even though they could use these same Abs to attach to or phagocytize inert particles. Under conditions of Ag excess and low concentrations of Ab, monocytes bound primarily to the viral HA molecule, but showed some recognition of the viral immune complexes. This dual binding did not increase monocyte adherence to the infected epithelium above that observed with virus alone. These findings indicate that neutralizing concentrations of IgA or IgG Abs, the predominant Abs found in the upper and lower respiratory tract, respectively, do not augment and can prevent monocyte recognition of influenza virus. This suggests that in vivo monocytes must use a non-Fc-mediated mechanism to adhere to and phagocytize these immune complexes.

Virus-neutralizing antibodies of immunoglobulin G (IgG) but not of IgM or IgA isotypes can cure influenza virus pneumonia in SCID mice

The ability of monoclonal antibodies (MAbs) to passively cure an influenza virus pneumonia in the absence of endogenous T- and B-cell responses was investigated by treating C.B-17 mice, homozygous for the severe combined immunodeficiency (SCID) mutation, with individual monoclonal antiviral antibodies 1 day after pulmonary infection with influenza virus PR8 [A/PR/8/34 (H1N1)]. Less than 10% of untreated SCID mice survived the infection. By contrast, 100% of infected SCID mice that had been treated with a single intraperitoneal inoculation of at least 175 micrograms of a pool of virus-neutralizing (VN+) antihemagglutinin (anti-HA) MAbs survived, even if antibody treatment was delayed up to 7 days after infection. The use of individual MAbs showed that recovery could be achieved by VN+ anti-HA MAbs of the immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgG3 isotypes but not by VN+ anti-HA MAbs of the IgA and IgM isotypes, even if the latter were used in a chronic treatment protocol to compensate for their shorter half-lives in vivo. Both IgA and IgM, although ineffective therapeutically, protected against infection when given prophylactically, i.e., before exposure to virus. An Fc gamma-specific effector mechanism was not an absolute requirement for antibody-mediated recovery, as F(ab')2 preparations of IgGs could cure the disease, although with lesser efficacy, than intact IgG. An anti-M2 MAb of the IgG1 isotype, which was VN- but bound well to infected cells and inhibited virus growth in vitro, failed to cure. These observations are consistent with the idea that MAbs of the IgG isotype cure the disease by neutralizing all progeny virus until all productively infected host cells have died. VN+ MAbs of the IgA and IgM isotypes may be ineffective therapeutically because they do not have sufficient access to all tissue sites in which virus is produced during influenza virus pneumonia.

Induction of immune response to influenza virus with anti-idiotypic antibodies

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against five monoclonal antibodies (MAbs) specific for different antigenic sites on the hemagglutinin (HA) of influenza virus Mem71H-BelN (H3N1) [A/Memphis/1/71 (H3N2) x A/Bel/42 (H1N1)]. Each of the anti-Id sera was directed predominantly towards a unique (private) idiotype of the immunizing MAb, none of the five idiotypes being detectable in pooled BALB/c antisera against Mem71H-BelN virus or on most other anti-HA MAbs tested. Partial idiotypic sharing was observed, however, between certain MAbs, from different mice, having the same or similar epitope specificity for HA. When used as immunogens in BALB/c mice, two of the five anti-Id preparations induced antibodies that reacted with Mem71H-BelN virus and displayed neutralizing activity. Mice of other inbred strains responded similarly, indicating that the response was not genetically restricted by the Igh locus. From their pattern of reactivity with mutants of Mem71H-BelN virus with known single amino acid substitutions in the HA molecule, the antiviral antibodies elicited by anti-Id antibodies were shown to be directed to the same antigenic site on A/Memphis/1/71 HA as the original immunizing MAb (site A or site E, respectively). However, several of these antisera were shown to contain additional distinct subpopulations of antibodies specific for heterologous influenza A virus strains, either of the H3 subtype or of a different HA subtype (H1 or H2). Since the induction of antibodies to HA of different subtypes is not a feature of the antibody response to influenza virus itself, their induction by anti-Id antibodies merits further investigation.