Carolynne Stanley

An inhibition enzyme immunoassay for estimating relative antibody affinity and affinity heterogeneity

A method to measure the relative affinity of antibodies using an inhibition enzyme immunoassay is described. It is validated using monoclonal antibodies of defined affinity characteristics and by comparison with conventional methods of affinity measurement. The method allows measurement of the relative affinity of low levels of antibody, and the calculation of an empirical estimate of the heterogeneity of affinity in antibody populations.

The influence of previous exposure to environmental mycobacteria on the interferon-gamma response to bacille Calmette-Guérin vaccination in southern England and northern Malawi.

We report a large study of the effect of BCG vaccination on the in vitro 6‐day whole blood interferon‐gamma (IFN‐γ) response to antigens from eight species of mycobacteria among schoolchildren in south‐eastern England, where bacille Calmette–Guérin (BCG) vaccination is highly protective against pulmonary tuberculosis, and among young adults in northern Malawi, where BCG vaccination is not protective. In the UK children, BCG induced an appreciable increase in IFN‐γ response to antigens from most species of mycobacteria. The degree of change was linked to the relatedness of the species to Mycobacterium bovis BCG, and provides further evidence of the cross‐reactivity of mycobacterial species in priming of the immune system. IFN‐γ responses to purified protein derivatives (PPDs) from M. tuberculosis and environmental mycobacteria were more prevalent in the Malawian than the UK group prior to vaccination; BCG vaccination increased the prevalence of responses to these PPDs in the UK group to a level similar to that in Malawi. There was no evidence that the vaccine‐induced change in IFN‐γ response was dependent upon the magnitude of the initial response of the individual to environmental mycobacteria in the United Kingdom or in Malawi. These observations should assist the development and interpretation of human clinical trials of new vaccines against M. tuberculosis in areas of both low and high exposure to environmental mycobacteria.

Influence of Antibody affinity on the performance of different antibody assays

The effect of antibody affinity on the performance of 5 commonly used assays was studied. The assays used were measurement of antigen binding capacity in a Farr type assay, haemagglutination, solid-phase radioimmunoassay (SP-RIA), solid-phase ELISA and precipitation. The first 4 assays were all more sensitive for high affinity antibodies. Precipitation was not related to affinity, suggesting that factors secondary to antigen-antibody binding may be more important in determining the level of precipitate formation. The effect of epitope density of the antigen was also investigated in the SP-RIA and ELISA. Affinity dependence was more marked when antigen of low epitope density was used and this dependence could be reduced in the ELISA by choosing a low OD endpoint. Thus, the most reliable way to estimate antibody content by the ELISA may be to determine a low OD endpoint titre against antigen of high epitope density. When epitope density per molecule cannot be increased, an alternative approach to the problem is to increase epitope density by covalent coupling of antigen to the solid-phase rather than by adsorption.

Immune responses in mice following immunization with chimeric synthetic peptides representing B and T cell epitopes of measles virus proteins.

The immunogenicity of chimeric peptides produced by collinear synthesis to contain both T and B cell epitopes from the fusion protein and the haemagglutinin of measles virus was studied in mice. The T cell epitope used was from the fusion protein (residues 288 to 302), which has been shown to be promiscuous in its binding to mouse major histocompatibility complex molecules. This epitope was coupled by (i) a glycine-glycine spacer to a B cell epitope from the fusion protein (residues 404 to 414) and (ii) either its amino or carboxy terminus to a neutralizing antibody epitope from the haemagglutinin (residues 188 to 199). The results obtained show that such chimeric peptides can indeed function as complete immunogens in a range of mouse strains of different H-2 haplotype, and can induce the production of antibodies which bind to the fusion protein and to measles virus. Furthermore, it was shown that the orientation of the T cell epitope with respect to the B cell epitope had a significant effect upon the immunogenicity and antigenic specificity of the chimera. This work gives further support to the concept of rationally designed synthetic peptide vaccines.

The measurement of antibody affinity: a comparison of five techniques utilizing a panel of monoclonal anti-dnp antibodies and the effect of high affinity antibody on the measurement of low affinity antibody.

The affinities of 9 IgG1 monoclonal anti-dinitrophenol (DNP) antibodies for 3H-epsilon-DNP-L-lysine, 125I-HOP-DNP-L-lysine and 125I-DNP-human serum albumin (HSA) were determined. 3H-DNP-lysine was used in equilibrium dialysis and ammonium sulphate globulin precipitation assays; 125I-HOP-DNP-lysine was used in equilibrium dialysis and polyethylene glycol precipitation; and 125I-DNP5-HSA in the polyethylene glycol precipitation assay for affinity. The ranking order of the monoclonal antibodies in terms of affinity by the assays was significantly correlated. Of particular importance was the observation that the simple and widely applicable globulin precipitation assay utilizing a protein antigen produced affinity values which showed concordance with the least equivocal but cumbersome assay, equilibrium dialysis. Mixing of antibodies of high and low affinity demonstrated that even a low proportion of high affinity antibody had marked effect on measurements of the amount and affinity of a predominantly low affinity antibody preparation.

The affinity of IgG antibodies to gag p24 and p17 in HIV‐1‐infected patients correlates with disease progression

The affinity of anti‐gag antibody was studied for up to 9 years (1984–1993) in sera from 15 HIV‐1+ patients with haemophilia. On the basis of their 1993 clinical status patients were divided into two groups: (i) patients who remained asymptomatic (n= 9); and (ii) those who progressed to AIDS between late 1987 and 1993. The affinity constants of antibody for p24 and p17 were determined by a double isotope fluid‐phase radioimmunoassay; and the relationships between antibody affinity and titre, patient clinical course, CD4 cell counts and p24 antigenaemia were analysed. The affinity of p24‐ and p17‐specilic antibody was up to 100 times greater in asymptomatic patients than in patients who progressed to AIDS. Patients who developed AIDS either lost or failed to develop high‐affinity antibodies early in the infection. Asymptomatic patients maintained high‐affinity antibodies for several years; however, in some of these patients the affinity of anti‐p24 and p17 antibodies subsequently fell later in the study period. The presence of low‐affinity antibody and progressive reduction in the titre of specific antibody were earlier predictors of disease onset than CD4 cell counts. The failure to either develop or maintain high affinity gag‐specific antibody suggests an early impairment of T helper function in individuals who progressed to AIDS. The presence of antibody of high affinity could be essential in controlling virus replication and the onset of AIDS.

The effect of orientation of epitopes on the immunogenicity of chimeric synthetic peptides representing measles virus protein sequences

We have studied the influence of the orientation of T- and B-cell epitopes on the immunogenicity of chimeric synthetic peptides in terms of the ability of the T-cell epitope to provide help for the production of antibody to the B-cell epitope. A T-cell epitope from the fusion protein of measles virus (288-302), previously shown to act as a T-helper epitope in a panel of six inbred mouse strains, was co-linearly synthesized at either the amino- or carboxyl- terminus of a B-cell epitope from the haemagglutinin of the virus (188-199) with or without the inclusion of a glycine-glycine spacer. The four chimeric peptides were used to immunize a panel of five mouse strains and induced good anti-chimera antibody responses. In general, the chimeras in which the T-cell epitope was amino-terminal to the B-cell epitope induced antibodies which bound well to the B-cell epitope whereas the carboxyl-terminal orientation of the T-cell epitope with respect to the B-cell epitope failed to induce such antibody. These latter chimeras induced the production of antibodies which preferentially bound to the T-cell epitope. The inclusion of the gly-gly spacer in the chimeras did not enhance their immunogenicity nor did it increase antibody titres to the B-cell epitope. The affinity of the anti-peptide antibodies was markedly influenced by the orientation of the epitopes in the chimeras. The antibody elicited by the peptide in which the T-cell epitope was amino terminal to the B-cell epitope had significantly higher affinity for the B-cell epitope than that induced by immunization with the peptide in the reverse orientation.

Antibody responses to recombinant and plasma derived hepatitis B vaccines

The antibody response to hepatitis B surface antigen (anti-HBs) induced in 25 recipients of a recombinant hepatitis B vaccine derived from yeast was compared with that induced in 25 recipients of a vaccine prepared from hepatitis B surface antigen (HBsAg) derived from plasma. Anti-HBs affinity and specificity were compared using assays of antibody affinity with two different antigens, a complex of the major polypeptide of HBsAg (p25; molecular weight 25 000 daltons) covalently linked to its glycosylated form (gp30) prepared from native purified HBsAg, and a cyclical synthetic peptide representing amino acid residues 139-147 of the major polypeptide of HBsAg and known to represent a major part of an a determinant. There was no difference in anti-HBs affinity or molar antigen binding sites of the antibody measured with either antigen between the two groups. All subjects in both groups produced antibody that bound to the gp30/p25 complex antigen, whereas 22 of the recipients of the plasma derived vaccine compared with 24 of those receiving the yeast derived vaccine produced antibodies that bound to the cyclical synthetic peptide 139-147. These results support the finding of similar levels of anti-HBs, measured by commercial solid phase radioimmunoassay, in the two vaccine groups after three doses of vaccine. These results show no significant difference in the quantity, quality, or specificity of the anti-HBs response induced by the recombinant hepatitis B vaccine and the plasma derived hepatitis B vaccine.

Interferon-γ and skin test responses of schoolchildren in southeast England to purified protein derivatives from Mycobacterium tuberculosis and other species of mycobacteria

The immune responses of schoolchildren in southeast England to Mycobacterium tuberculosis and other species of mycobacteria were studied prior to vaccination with bacille Calmette‐Guérin (BCG). Data are presented for tuberculin (Heaf) skin test and interferon‐γ (IFN‐γ) responses to M. tuberculosis purified protein derivative (PPD), and IFN‐γ responses to PPDs from eight other environmental mycobacteria, measured in 424 schoolchildren (13–15 years of age). Responses to M. tuberculosis PPD were detected in 27% of schoolchildren by in vitro IFN‐γ response and in 20% by the Heaf test. IFN‐γ responses were more prevalent to PPDs from species of mycobacteria other than M. tuberculosis, predominantly those of the MAIS complex and M. marinum (45–60% responders). Heaf test and IFN‐γ responses were associated (P < 0·001) for M. tuberculosis, MAIS and M. marinum. These findings have implications for appropriate implementation of vaccination against tuberculosis.

Identification of immunological biomarkers which may differentiate latent tuberculosis from exposure to environmental nontuberculous mycobacteria in children.

ABSTRACT A positive gamma interferon (IFN-γ) response to Mycobacterium tuberculosis early secretory antigenic target-6 (ESAT-6)/culture filtrate protein-10 (CFP-10) has been taken to indicate latent tuberculosis (TB) infection, but it may also be due to exposure to environmental nontuberculous mycobacteria in which ESAT-6 homologues are present. We assessed the immune responses to M. tuberculosis ESAT-6 and cross-reactive responses to ESAT-6 homologues of Mycobacterium avium and Mycobacterium kansasii. Archived culture supernatant samples from children at 3 years post-BCG vaccination were tested for cytokine/chemokine responses to M. tuberculosis antigens. Furthermore, the IFN-γ responses to M. tuberculosis antigens were followed up for 40 children at 8 years post-BCG vaccination, and 15 TB patients were recruited as a control group for the M. tuberculosis ESAT-6 response in Malawi. IFN-γ enzyme-linked immunosorbent assays (ELISAs) on supernatants from diluted whole-blood assays, IFN-γ enzyme-linked immunosorbent spot (ELISpot) assays, QuantiFERON TB Gold-In Tube tests, and multiplex bead assays were performed. More than 45% of the responders to M. tuberculosis ESAT-6 showed IFN-γ responses to M. avium and M. kansasii ESAT-6. In response to M. tuberculosis ESAT-6/CFP-10, interleukin 5 (IL-5), IL-9, IL-13, and IL-17 differentiated the stronger IFN-γ responders to M. tuberculosis ESAT-6 from those who preferentially responded to M. kansasii and M. avium ESAT-6. A cytokine/chemokine signature of IL-5, IL-9, IL-13, and IL-17 was identified as a putative immunological biosignature to differentiate latent TB infection from exposure to M. avium and M. kansasii in Malawian children, indicating that this signature might be particularly informative in areas where both TB and exposure to environmental nontuberculous mycobacteria are endemic.