HIV hyperimmune globulin or intravenous immune globulin inhibits response to an HIV vaccine.

The murine model was developed to assess the effects of maternally transferred HIV hyperimmune globulin or human intravenous immune globulin on the immunization of the offspring at 18-21 days of age with rgp 120SF2-complete Freund's adjuvant. Either HIV hyperimmune globulin or intravenous immune globulin was administered intraperitoneally to post-partum BALB/c mice and was transferred via milk to the offspring. Both HIV hyperimmune globulin and intravenous immune globulin inhibited the offspring anti-rgp 120SF2 IgG response to the vaccine. The HIV hyperimmune globulin inhibition persisted for 28 days after immunization while the intravenous immune globulin inhibition was still present at 63 days after immunization. In addition, the intravenous immune globulin had a more generalized immunosuppressive effect, inhibiting the IgG response to both rpg 120SF2 and an additional protein antigen, hen egg-white lysozyme. Effects of maternal or exogenously administered pre-existing antibody, including control antibodies (intravenous immune globulin), on the newborn response to HIV and other vaccines must be carefully evaluated when vaccine studies proceed in newborns.

Direct binding of the MHC class I molecule H-2Ld to CD8: interaction with the amino terminus of a mature cell surface protein.

MHC class I molecules (MHC-I) display peptides from the intracellular pool at the cell surface for recognition by T lymphocytes bearing alphabeta TCR. Although the activation of T cells is controlled by the interaction of the TCR with MHC/peptide complexes, the degree and extent of the activation is influenced by the binding in parallel of the CD8 coreceptor with MHC-I. In the course of quantitative evaluation of the binding of purified MHC-I to engineered CD8, we observed that peptide-deficient H-2Ld (MHC-I) molecules bound with moderate affinity (Kd = 7.96 x 10(-7) M), but in the presence of H-2Ld-binding peptides, no interaction was observed. Examination of the amino terminal sequences of CD8alpha and beta chains suggested that H-2Ld might bind these protein termini via its peptide binding cleft. Using both competition and real-time direct assays based on surface plasmon resonance, we detected binding of empty H-2Ld to synthetic peptides representing these termini. These results suggest that some MHC molecules are capable of binding the amino termini of intact cell surface proteins through their binding groove and provide alternative explanations for the observed binding of MHC molecules to a variety of cell surface receptors and coreceptors.

Phosphorylation of extracellular domains of T-lymphocyte surface proteins. Constitutive serine and threonine phosphorylation of the T cell antigen receptor ectodomains.

The extracellular accumulation of ATP after activation of T-lymphocytes, as well as the presence of ecto-protein kinases in these cells, led us to propose that T cell surface receptors could be regulated through the reversible phosphorylation of their extracellular domains (ectodomains). Here, in a model system, we used T cell transfectants which express T cell antigen receptor chains lacking intracellular and transmembrane protein domains and 32Pi metabolic labeling of cells to definitively demonstrate phosphorylation of ectodomains of T cell surface proteins. We show that alphabetaTCR ectodomains were phosphorylated intracellularly and constitutively on serine and threonine residues and were then expressed on the T cell surface in phosphorylated form. TCR ectodomains also could be phosphorylated at the cell surface when extracellular [gamma-32P]ATP or [gamma-32P]GTP were used as phosphate donors with the same cells. Consensus phosphorylation sites for serine and threonine protein kinases were found to be strongly evolutionary conserved in both alpha and beta TCR chains constant regions. These results are consistent with the hypothesis, where T cell surface proteins which are phosphorylated intracellularly on their ectodomains, could subsequently be expressed at the cell surface and then be reversibly modified by ectoprotein phosphatase(s) and by ectokinase(s). Such modifications may change T cells cognate interactions by, e.g. affecting TCR-multimolecular complex formation and antigen binding affinity. It is suggested that alphabetaTCR ectodomain phosphorylation could serve as a potential mechanism for regulation of alphabetaTCR-mediated T-lymphocytes response.

Maternal monoclonal antibody to the V3 loop alters specificity of the response to a human immunodeficiency virus vaccine.

The effect of maternally transferred monoclonal antibody (MAb) on the offspring antibody response to rgp120SF2 was examined in a murine model. Two MAbs were studied: MAb 83.1, which recognizes a determinant in the V3 loop of gp120 from human immunodeficiency virus-1 (HIV-1) SF2, and MAb 26.2D3, which recognizes a conserved N-terminal region of gp120 from HIV-1SF2. Offspring were immunized at 18-21 days of age with 100 micrograms of rgp120SF2 in complete Freund's adjuvant. Offspring immunized in the presence of preexisting MAb 83.1 but not MAb 26.2D3 demonstrated inhibition of the IgG anti-V3 response. The total IgG anti-rgp120SF2 response was not affected by preexisting MAb. Since newborns at risk for HIV may be immunized in the presence of maternal or administered anti-HIV antibody, alternative strategies may be required to circumvent inhibition of the infant's epitope-specific response to HIV immunization by preexisting antibody.

Studying interactions involving the T-cell antigen receptor by surface plasmon resonance.

T-lymphocyte activation is initiated by the interaction of the alpha beta TCR with a complex consisting of a class I or class II MHC-encoded molecule and an antigenic peptide, displayed on the surface of an antigen-presenting cell. Real-time binding measurements using surface plasmon resonance have revealed kinetic and equilibrium parameters for the interactions between purified MHC molecules and peptides, between TCR and MHC-peptide complexes, and between TRC and superantigens. The MHC-peptide interaction is characterized by its high affinity and long half-life, the TCR-MHC/peptide interaction by its low affinity and short half-life, and the TCR-superantigen interaction by its low-to-moderate affinity, which is dependent on the particular superantigen involved. The consistent finding is that both MHC-peptide complexes and superantigens interact with TCR with a low affinity attributable to rapid dissociation. That an MHC-peptide complex that encounters a single TCR only briefly can still deliver the necessary activation signals offers a mechanistic conundrum for which several solutions have been proposed.

Inhibition of the offspring anti-recombinant gp120 antibody response to a human immunodeficiency virus vaccine by maternal immunization in a murine model.

A murine model was developed for assessing the effects of passively transferred polyclonal maternal anti-gp120 antibodies on the subsequent immunization of the offspring with recombinant gp120SF2 in complete Freund's adjuvant (rgp120SF2-CFA). Adult female BALB/c mice were immunized with rgp120SF2-CFA 6 weeks before mating. The 3-week-old offspring were subsequently immunized with the same vaccine and followed for 9 weeks. Both the total IgG anti-rgp120SF2 and the anti-V3 IgG antibody response to vaccine were inhibited in the experimental animals. The total IgG anti-rgp120SF2 response was < 20% of the control response (P < .001) 9 weeks after immunization. Anti-V3 antibody was also decreased. As vaccine studies begin in infants, the effects of preexisting antibody on the infant response to human immunodeficiency virus vaccines must be considered.

Lack of strict correlation of functional sensitization with the apparent affinity of MHC/peptide complexes for the TCR.

We describe a comprehensive analysis of the effect of avidity of TCR-MHC/peptide interaction on activation of the (p2Ca). In study, monosubstituted variants of p2Ca were used and assessed for binding to purified H-2Ld, binding of H-2Ld/peptide complexes to sTCR, and ability to activate 2C cells to two independent effector functions. Among the > 20 variants analyzed, functional activity of most peptides that bound the MHC well correlated with the strength of interaction of MHC/peptide complexes with sTCR. However, with some variants, a clear discordance between the apparent TCR-MHC/peptide affinity and biologic function was observed, demonstrating that the former cannot always be gauged by the latter. In the case of L4 peptide (phenylalanine at position 4 substituted with leucine), peptide/MHC complexes showed no detectable binding to sTCR, indicating a 10-fold or greater decrease in affinity. Nevertheless, this peptide sensitized target cells for lysis at a level equivalent to the parental peptide. A clearer understanding was revealed by studying the extent to which activation by variant peptides was dependent on CD8. Our data indicate that resistance to anti-CD8 mAb blocking correlates with strong binding affinity between sTCR and MHC/peptide complexes. These data suggest that, for the activation of CTL function, the absolute level of intrinsic affinity of TCR for MHC/peptide ligand is not a single critical determinant, but rather, that activation is governed by the compound influence of several factors, which ensures a minimum threshold of intracellular triggering is reached to elicit the response.

Measuring interactions of MHC class I molecules using surface plasmon resonance.

To examine the molecular interactions between major histocompatibility complex (MHC)-encoded molecules and peptides, monoclonal antibodies (mAbs), or T cell receptors, we have developed model systems employing genetically engineered soluble MHC class I molecules (MHC-I), synthetic peptides, purified mAbs, and engineered solubilizable T cell receptors. Direct binding assays based on immobilization of one of the interacting components to the dextran modified gold biosensor surface of a surface plasmon resonance (SPR) detector have been developed for each of these systems. The peptide binding site of the MHC-I molecule can be sterically mapped by evaluation of a set of peptides immobilized through the thiol group of cysteine substitutions at each peptide position. Kinetic binding studies indicate that the MHC-I/peptide interaction is characterized by a low to moderate apparent kass (approximately 5000-60000 M-1 s-1) and very small kdis (approximately 10(-4)-10(-6) s-1) consistent with the biological requirement for a long cell surface residence time to permit engagement with T cell receptors. Several mAb directed against different MHC-I epitopes were examined, and kinetic parameters of their interaction with MHC molecules were determined. These showed characteristic moderate association rate constants and moderate dissociation rate constants (kass approximately 10(4)-10(6) M-1 s-1 and kdis approximately 10(-2)-10(-4) s-1), characteristic of many antibody/protein antigen interactions. The interaction of an anti-idiotypic anti-TCR mAb with its purified cognate TCR was of moderate affinity and revealed kinetic binding similar to that of the anti-MHC mAbs. The previously determined interaction of a purified T cell receptor with its MHC-I/peptide ligand is characterized by kinetic constants more similar to those of the antibody/antigen interaction than of the MHC-I/peptide interaction, but is remarkable for rapid dissociation rates (apparent kdis approximately 10(-2) s-1). Such binding studies of reactions involving the MHC-I molecules offer insight into the mechanisms responsible for the initial specific events required for the stimulation of T cells.

T cell receptor-MHC class I peptide interactions: affinity, kinetics, and specificity.

The critical discriminatory event in the activation of T lymphocytes bearing alpha beta T cell receptors (TCRs) is their interaction with a molecular complex consisting of a peptide bound to a major histocompatibility complex (MHC)-encoded class I or class II molecule on the surface of an antigen-presenting cell. The kinetics of binding were measured of a purified TCR to molecular complexes of a purified soluble analog of the murine MHC class I molecule H-2Ld (sH-2Ld) and a synthetic octamer peptide p2CL in a direct, real-time assay based on surface plasmon resonance. The kinetic dissociation rate of the MHC-peptide complex from the TCR was rapid (2.6 x 10(-2) second-1, corresponding to a half-time for dissociation of approximately 27 seconds), and the kinetic association rate was 2.1 x 10(5) M-1 second-1. The equilibrium constant for dissociation was approximately 10(-7) M. These values indicate that TCRs must interact with a multivalent array of MHC-peptide complexes to trigger T cell signaling.

Comparison of naturally acquired and vaccine-induced antibodies to Haemophilus influenzae type b capsular polysaccharide.

The objective of this study was to assess qualitative differences in the types of Haemophilus influenzae type B (Hib) capsular polysaccharide (polyribosylribitol phosphate [PRP]) antibodies induced in children 15 to 27 months of age by (i) natural exposure, (ii) PRP vaccine, and by (iii) PRP-diphtheria toxoid conjugate vaccine, (iv) PRP-group B Neisseria meningitidis outer membrane vesicle conjugate vaccine, and (v) Haemophilus type B oligosaccharide conjugate vaccine (HbOC). The highest levels of total Hib-PRP antibody measured by radioimmunoassay and immunoglobulin G (IgG) measured by enzyme-linked immunosorbent assay were seen after HbOC immunization. IgG1 Hib-PRP antibodies predominated in all groups, and there were no differences between the groups in the proportion of IgG and IgA Hib-PRP antibodies. However, the proportions of IgM differed significantly by group. The highest proportions of IgM occurred in naturally acquired antibody and after PRP vaccine, and the lowest proportion occurred after HbOC vaccine. IgG light-chain V kappa type alpha PRP antibody was present in all groups, and the level correlated with the total IgG Hib-PRP antibody level. Therefore, HbOC induced the highest concentrations of V kappa II type alpha PRP antibody, and the naturally acquired antibody group had the lowest levels. IgG light-chain V kappa III antibody levels were also highest in the HbOC group, but there was no correlation between V kappa III antibody levels and total amount of IgG Hib-PRP antibody. These data demonstrate qualitative differences in the antibody repertoires induced by natural exposure, the Hib-PRP vaccine, and each of the different Hib conjugate vaccines. We doubt that there are major differences in the protection afforded by these different antibody repertoires, because these differences do not appear to correlate with differences in protective efficacy in older children.

Maternal HEL immunization has no lasting effects on the immune response of offspring to immunization with hen egg-white lysozyme.

The effect of prior maternal immunization on the murine offspring response to subsequent immunization with hen egg-white lysozyme was examined. Adult female A/J mice were immunized with 100 micrograms HEL-CFA intraperitoneally 10-27 weeks before conception. The offspring of these experimental female mice were then immunized with HEL-CFA at differing ages. Suppression of the anti-HEL IgG B cell response was observed when the offspring were immunized prior to 3 weeks of age when high levels of maternal antibody were still present. Older offspring, more than 8 weeks of age, were immunized with HEL-CFA to determine if exposure to maternal immunoglobulin early in ontogeny had primed or altered the offspring response to HEL. At this age, suppressive effects of transferred maternal antibody were no longer evident. Priming was not detected in the offspring as judged by the total magnitude of the anti-HEL antibody response or the kinetics of the response when experimental and age-matched control offspring were examined. Furthermore, qualitative differences in the response as evidenced by IgG vs IgM content and fine specificity of the response (primary vs secondary antibody) were not observed. No evidence was found to suggest that exposure to polyclonal maternal anti-HEL antibody had primed the offspring for a more efficient or qualitatively different response to immunization with the protein antigen HEL. After maternal antibody levels decreased, the offspring response was similar to that of controls, suggesting that the response had not been permanently altered by the prior exposure early in ontogeny to polyclonal maternal antibody.