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W Gerhard

Publications and source records attributed to W Gerhard.

At least 73 records · Page 4Linked to original sources
PubMed1985

Longitudinal analysis of three intrathecally produced immunoglobulin subpopulations in an MS patient.

Four murine anti-idiotypic (a-Id) hybridoma antibodies were produced against immunoglobulins (Ig) present in the cerebrospinal fluid (CSF) obtained from an MS patient 2 mo after the onset of disease. The four a-Id antibodies were shown to delineate idiotopes present on three distinct Ig subpopulations designated ID-19, ID-40, and ID-97. All three Ig subpopulations were produced in part by intrathecally localized B cells, together making up approximately 5% of the total CSF-Ig 2 mo after the onset of disease. Longitudinal analysis of the concentration of these Ig subpopulations in CSF showed that two subpopulations, ID-40 and ID-97, exhibited a regular relation to the clinical course of the disease, i.e., were decreased (ID-40) or increased (ID-97) in the first CSF sample obtained after two consecutive exacerbations. Screening of sera from 52 optic neuritis patients and 51 heterologous MS patients revealed that one MS patient's serum contained an Ig subpopulation that was idiotypically cross-reactive with ID-97. So far, screening of these Ig subpopulations for reaction with several viruses (measles, parainfluenza type 1, influenza type A, cytomegalovirus, herpes simplex virus, rubella virus, poliovirus, murine encephalomyelitis viruses, and reovirus) and myelin basic protein has failed to reveal their antigen specificities.

Adult↗
PubMed1985

Murine TH response to influenza virus: recognition of hemagglutinin, neuraminidase, matrix, and nucleoproteins.

BALB/c mice were primed with type A influenza virus by footpad injection or by aerosol infection with PR8 [A/PR/8/34-(H1N1)]. Isolated T cells from draining lymph nodes were then tested for their proliferation in the presence of purified viral proteins hemagglutinin, neuraminidase, matrix, and nucleoprotein. Significant responses [( 3H]thymidine incorporation) were seen against each of the four proteins after either priming scheme. When helper T (TH) cell clones were isolated by hybridoma formation from two different strains of mice, responsiveness (interleukin 2 production) towards each protein was against apparent. Of 12 virus-specific T cell hybridomas isolated, four responded to matrix, three to nucleoprotein, one to neuraminidase, three to hemagglutinin, and one cell was of undefined specificity. Each hybridoma was also tested for recognition of the HK virus [A/Hong Kong/1/68-(H3N2)], which differs in subtype from the priming strain. All matrix-specific cells, two nucleoprotein-specific cells, and the cell of undefined specificity were cross-reactive with HK virus. H1-subtype specificity was seen for all hemagglutinin and neuraminidase-specific cells and one of the three nucleoprotein-specific cells. Because many virus-immune TH cells recognize antigenically variable determinants, a significant fraction of TH cell function may be lost after virus evolution. When selecting priming schemes for long-term immunization against influenza, the isolated enhancement of TH cells recognizing conserved determinants on matrix and nucleoprotein may therefore be considered.

Animals↗
PubMed1984

Generation of antibody diversity in the immune response of BALB/c mice to influenza virus hemagglutinin.

We have examined the amino-terminal sequence of the kappa light chains of a set of monoclonal antibodies specific for one of the major antigenic determinants (Sb) on the influenza virus PR8[A/PR/8/34(H1N1)] hemagglutinin molecule. This set was believed to be structurally related from earlier serological analysis that typed these kappa chains as members of the variable (V) region V kappa 21 group [ Staudt , L. M. & Gerhard , W. (1983) J. Exp. Med. 157, 678-704]. Our sequence analysis confirms and extends this conclusion; all examples of this set belong to a subgroup of the V kappa 21 group, V kappa 21C . A special feature of this set of kappa light chains is that all examples were derived from the same mouse (designated H36 ). This sequence analysis along with the characterization of gene rearrangements at the kappa light chain loci of these hybridomas is consistent with the idea that certain members of this set are the progeny of one or two lymphocytes. Because of this potential clonal relationship, we can reach several conclusions about the diversity observed among these kappa light chains: (i) the diversity is due to somatic mutation, (ii) somatic mutations occur sequentially and accumulate in the first complementarity-determining region, and (iii) the extent of somatic variation in this sample is high, suggesting a somatic mutation rate of about 10(-3) per base pair per generation.

Amino Acid Sequence↗
PubMed1984

Characterization of the murine TH response to influenza virus hemagglutinin: evidence for three major specificities.

Several hemagglutinin (HA)-specific T cell hybridomas were generated from five PR8 influenza virus-primed BALB/c mice by fusion with the thymoma BW5147 and were tested for IL 2 (interleukin 2) production in response to a large panel of serologically distinct influenza viruses and HA-protein fragments. The hybridomas (provisionally categorized in the helper T cell lineage) could be placed into three major specificity groups based on their reactivity patterns toward 43 antibody-selected PR8 virus mutants that had been shown previously to differ from parental PR8 virus by defined (usually single) amino acid substitutions in the HA1 polypeptide. Three hybridomas with specificity 1 failed to respond to the PR8 mutant RV6 (Glu115----Lys), two with specificity 2 failed to respond to PR8 mutant DV4 (Ser136----Pro), and two with specificity 3 responded to all antibody-selected PR8 mutants without exception. The three reactivity patterns could be used along with further biochemical evidence to demonstrate the presence of three distinct T cell determinants on the HA1 polypeptide. Priming of mice with one of these T cell determinants in the form of a synthetic peptide induced a T cell subset able to proliferate in vitro in response to HA and intact PR8 virus. Further analysis of the T cell hybridomas for reaction with 17 natural influenza virus isolates of the H1 subtype revealed additional differences in fine nominal antigen specificity among these T cells. However, two pairs of T cells whose members were isolated from different mice exhibited indistinguishable fine nominal antigen specificities. The latter analysis showed that natural antigenic drift (i.e., accumulation of point mutations) in the HA occurring between the years 1934 and 1957 and again between 1977 and 1980 altered two T cell determinants but left one T cell determinant unchanged. The shifts from H1 to H2 or H3 subtypes resulted in the alteration of all three T cell determinants. The present findings suggest that 1) immunization with influenza virus induces HA-specific T cells that recognize determinants different from those seen by anti-HA antibodies, and 2) the HA-specific T cell repertoire of adult virus-primed BALB/c mice is more restricted than the B cell repertoire.

Animals↗
PubMed1983

Influenza virus site recognized by a murine helper T cell specific for H1 strains. Localization to a nine amino acid sequence in the hemagglutinin molecule.

The functional helper T cell line Vir-2, derived from a PR8 (H1N1) influenza virus-immunized BALB/c mouse, proliferates in response to syngeneic antigen-presenting cells and naturally occurring strains of subtype H1 human influenza virus from 1934-1957 and 1977-1980 isolates. A conserved region of the hemagglutinin molecule around amino acid position 115 in the heavy chain (HA1) was implicated as being important in this recognition by the lack of stimulatory activity associated with a glutamic acid to lysine substitution at position 115 in the laboratory mutant RV6, derived from wild-type PR8. Characterization of the stimulatory determinant on the wild-type hemagglutinin molecule was then undertaken using cleavage products and synthetic peptides. Vir-2 cells recognized the reduced and alkylated purified HA1 of PR8 virus, and this reactivity was retained after cleavage at methionine and tryptophan residues. High-pressure liquid chromatography separation of cleavage fragments indicated that a short sequence of the HA1 containing residue 115 was being recognized. This recognition was localized to a nine amino acid segment (positions 111-119) by assaying stimulation with synthetic peptide homologues of different lengths from that region. As with native hemagglutinin, Vir-2 cells responded to active peptides when presented by H-2d but not H-2k antigen-presenting cells.

Amino Acid Sequence↗
PubMed1983

Generation of antibody diversity in the immune response of BALB/c mice to influenza virus hemagglutinin. I. Significant variation in repertoire expression between individual mice.

The paratypic and idiotypic diversity of the BALB/c antibody response to the hemagglutinin (HA) of the influenza A/PR/8/34 virus (PR8) was investigated using a panel of 125 anti-HA hybridoma antibodies derived from 14 BALB/c mice. The paratypic diversity, as assessed by a fine specificity analysis using 51 related influenza viruses, was extensive: 104 distinct paratopes were observed. In three instances, antibodies with indistinguishable paratopes were isolated from two individual mice. A minimum estimate of the size of the adult BALB/c anti-HA paratypic repertoire, calculated from these data, is 1,500. The generation of this diverse repertoire was studied by screening the anti-HA hybridoma panel for the presence of idiotypes (Id) that are markers for variable (V) region sequences derived from related germ line V genes. Three cross-reactive Id (IdX) that are markers for the V(k)21C, V(k)21B, and V(k)21A, D, E, or F L chain subgroups were found, respectively on 16, 1, and 10 anti-HA hybridoma antibodies derived from seven individual BALB/c mice. Thus, the V(k)21 IdX(+) hybridomas constitute 22 percent of the anti-HA hybridoma panel. The V(k)21 IdX are also present on 8.6 percent of K-bearing immunoglobulin in normal BALB/c serum. This suggests that the V(k)21 group is used preferentially in the BALB/c anti-HA immune response. The generation of the anti-HA repertoire was further studied using large panels of anti-HA hybridomas derived from two individual adult BALB/c mice. Anti-idiotypic antisera were raised in rabbits against individual hybridomas from each mouse. One anti-Id serum defined a family of four idiotypically and paratypically related, but not identical, antibodies from mouse 36, which represented 31 percent of the hybridoma antibodies isolated from this mouse. None of the 112 anti-HA hybridoma antibodies derived from 13 other individual mice showed idiotypic cross-reactivity. Furthermore, this Id could not be detected in anti-PR8 antisera from 75 individual BALB/c mice. Another anti-Id serum defined a family of 27 idiotypically related antibodies from mouse 37, which represented 50 percent of the hybridoma antibodies isolated from this mouse. Only 1 of the 71 hybridoma antibodies isolated from 13 other individuals was idiotypically cross-reactive. These results demonstrate that individual adult BALB/c mice express paratypically and idiotypically distinct antibody repertoires to the HA of influenza virus PR8. Based on these observations, we suggest that somatic mutation plays an important role in the generation of the adult anti-HA repertoire. Mechanisms that could account for differences in repertoire expression among individual mice are discussed.

Animals↗
PubMed1983

Monoclonal antibodies to the hemagglutinin Sa antigenic site of a/pr/8/34 influenza virus distinguish biologic mutants of swine influenza virus.

The dimorphic L and H hemagglutinin mutants of A/NJ/11/76(H1N1) (swine) influenza virus differ pleiotropically in their replication and virulence characteristics and in their antigenicity. L mutants replicate less well in chicken embryos and Madin-Darby canine kidney cells and are more infective for swine than are H mutants. L and H mutants are not antigenically distinguishable in cross-neutralization tests with homotypic antisera, but they can be identified with certain heterotypic heterogeneous antisera. The present studies demonstrate that two monoclonal antibodies (Sa-5 and Sa-13) to the Sa antigenic site of the hemagglutinin of A/PR/8/34H1N1 influenza virus react with mutants and viral reassortants containing the H hemagglutinin in radioimmunoassay, neutralization, and hemagglutination-inhibition tests but to a lesser degree or not at all with L mutants and reassortants. Conversely, monoclonal antibody (9C8) to the L mutant does not react with H mutants. L to H and H to L revertants, whether or not selected with monoclonal antibody, demonstrate concomitant change in biological and antigenic phenotype. Reactivity of H mutants with Sa monoclonal antibodies localizes the mutational site to a position on the hemagglutinin near the receptor binding site--a position in which single amino acid changes could readily influence both antigenic and biologic activity.

Antibodies, Monoclonal↗
PubMed1983

Monoclonal anti-hemagglutinin antibodies detect irreversible antigenic alterations that coincide with the acid activation of influenza virus A/PR/834-mediated hemolysis.

Exposure of influenza virus to an acidic environment, which is known to be required for viral fusion and hemolysis, has recently been shown to induce a conformational change in the hemagglutinin molecule. In the present study, we examined the effects of acid incubation on the antigenicity, biological activity, and morphology of influenza virus A/PR/8/34 (H1N1). Incubation of PR8 virus at pH 5 in the absence of erythrocytes resulted in a rapid and irreversible loss of viral hemolytic activity and infectivity. Apart from a less distinct appearance of the viral surface projections and slight damage to the envelope structure, acid incubation did not result in gross morphological changes in the viral architecture. The acid-induced change could be detected in the form of greatly increased or decreased binding of many monoclonal antibodies directed to each of the four major antigenic regions of the hemagglutinin. Triggering of viral hemolytic activity and antigenic alterations was similarly pH dependent. In addition, the different pH dependencies of egg-grown and trypsin-treated MDCK-grown viruses coincided with an analogous pH dependence of the antigenic alterations that were observed with these viruses. These observations are compatible with the idea that some of the anti-hemagglutinin antibodies detect conformational changes in the hemagglutinin which are required for the initiation of fusion and hemolysis.

Antibodies, Monoclonal↗
PubMed1983

The recognition specificity of a murine helper T cell for hemagglutinin of influenza virus A/PR/8/34.

A T cell line was established in long-term tissue culture from spleen cells of BALB/c mice immunized with influenza virus PR8 (A/PR/8/34(H1N1)) by continuous restimulation with PR8 virus and syngeneic x-irradiated spleen cells. This T cell line and clones derived from it have now been propagated for over 1 yr "in vitro". The T cell line and the clones, upon stimulation with Con A in the absence of antigen and irradiated spleen cells, as upon stimulation in the presence of antigen and adherent cells, produce T cell growth factors, B cell replication and maturation factors, and colony-stimulating factors, as tested by restimulation of proliferation of a clone of cytolytic murine T cells, of lipopolysaccharide-activated B cell blasts, and by macrophage/granulocyte colony formation of murine bone marrow cells, respectively. The T cell line and the clones help syngeneic B cells in the presence of syngeneic irradiated spleen cells and PR8 virus to proliferate and mature into clones of cells secreting virus-specific antibodies. In the presence of PR8 virus and of a bystander antigen, such as sheep red cells, B cells specific for this bystander antigen are also induced. Adoptive transfer of the T cells i.v. into syngeneic nu/nu BALB/c mice enable the recipient mice to produce, upon challenge with PR8, virus-specific antibodies and to clear virus infection of the lung. The virus-specific T cell line and the clones are restricted by H-2 antigens of the d-haplotype, probably by Iad, and recognize a determinant on the hemagglutinin (HA) molecule of PR8 virus. Fifty to 100 fmol of virus-associated or free HA suffice for stimulation as measured by proliferation assays. The stimulating determinant is present on the HA of all natural virus isolates of the H1 subtype, is absent from virus isolates of the H2 and H3 subtypes, and is abolished if glutamic acid at position 115 of the HA1 polypeptide of PR8 is replaced by lysine.

Animals↗
PubMed1982

The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype).

We have constructed an operational antigenic map of the hemagglutinin of influenza virus A/PR/8/34, which indicates the presence of five immunodominant antigenic regions exhibiting various degrees of operational linkage. These sites have been located by the identification of changed amino acid residues in mutant viruses that are antigenically altered at each site. Comparison of the antigenic features with the three-dimensional structure of the H3 subtype hemagglutinin shows that the antigenic sites correspond to four topographically distinct regions of the surface of the protein. One of the sites is formed when two regions that are widely separated in the hemagglutinin monomer associate in the assembled trimer. The location of the sites relative to those proposed for the H3 subtype hemagglutinin suggests that carbohydrate modulates the antigenicity of specific regions of the hemagglutinin.

Amino Acid Sequence↗
PubMed1982

Delineation of four antigenic sites on a paramyxovirus glycoprotein via which monoclonal antibodies mediate distinct antiviral activities.

Six hybridoma antibodies specific for the hemagglutinin-neuraminidase (HN) molecule of the parainfluenza type 1 virus strain 6/94 were used to demonstrate the existence of four distinct antigenic sites on the HN molecule. Three of the sites (A, B-C, D) are topologically nonoverlapping, because antibodies to these sites bind noncompetitively to the HN molecule. Two sites (B, C) are operationally nonoverlapping, because mutations in site B do not detectably modify the antigenic site C. Although antibodies to each site had similar potencies (activity per microgram of antibody) in hemagglutination inhibition tests, antibodies to sites A and C or D differed approximately 100-fold in their potency to neutralize virus. Also, the antibody to site A strongly inhibited viral neuraminidase activity, whereas antibodies to sites C and D (and to a lesser extent to site B) enhanced the neuraminidase activity. Lastly, only antibodies to sites C and D formed precipitates in Ouchterlony double diffusion against detergent-disrupted virus. Because all six anti-HN antibodies are of IgG isotype and exhibited similar avidity for HN, these findings suggest that the ability of anti-HN antibodies to interact with the viral protein and to alter viral functions is largely dependent on their fine specificity.

Animals↗