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Analysis of mumps vaccine failure by means of avidity testing for mumps virus-specific immunoglobulin G.

To characterize patients with mumps vaccine failure, avidity testing was performed with the Enzygnost Anti-Parotitis Virus/IgG kit using a single-dilution-6 M urea denaturation method. Five groups of patients were tested. Group 1 consisted of 29 patients with primary mumps infections; group 2 was 20 children and adults with a definite history of natural infection; group 3 was 7 patients with a recent mumps vaccination, 1 of whom developed parotid gland swelling and aseptic meningitis; group 4 was 14 patients with mumps vaccine failure; and group 5 was 6 patients with recurrent episodes of parotitis in addition to a history of vaccination. On the basis of the results of groups 1 and 2, an avidity of </=31% was determined to be low, and >/=32% was determined to be high. Avidity maturation from low to high appears to occur around 180 days after the acute illness. The results of group 3 showed that the vaccine-induced immunoglobulin G (IgG) had very low avidity. Among the 14 patients in group 4, 12 patients, including 7 with a positive IgM response, were diagnosed as having secondary vaccine failures. The results of group 5 suggested the possibility that the avidity of the mumps vaccine-induced IgG remains low or borderline. These results showed that secondary mumps vaccine failure occurs not infrequently, even among school age children under condition in which the vaccine coverage is low (i.e., 33% in our study population), and therefore, vaccinees are prone to be exposed to wild-type viruses. Avidity testing should provide information useful for the analysis of mumps virus infections.

Adolescent↗

Use of immunoglobulin g avidity to determine the course of disease in visceral and post-kala-azar dermal leishmaniasis patients.

In the present study, anti-Leishmania immunoglobulin G (IgG) avidity was used to estimate the approximate time of disease manifestation. Significant differences (P < 0.0001) were found between the levels of anti-rKE-16 IgG avidity in leishmaniasis patients with recent and chronic diseases. More than 76% of patients with an illness duration of less than 6 months had avidity of less than 70%, 94% of patients had less than 80% avidity, and all (100%) patients with illness of more than 6 months had avidity values higher than 70%. The study showed that avidity could successfully be used to pinpoint the duration of leishmaniasis.

Adult↗

Role of high-avidity binding of human neutrophil myeloperoxidase in the killing of Actinobacillus actinomycetemcomitans.

The binding of the neutrophil enzyme myeloperoxidase (MPO) to microbial surfaces is believed to be the first step in its microbicidal activity. The MPO-H2O2-Cl- system is responsible for most oxidative killing of Actinobacillus actinomycetemcomitans by human neutrophils. There appear to be three forms of MPO (MPO I, II, and III), all of which can kill this organism in the presence of H2O2 and chloride. In this study, we characterized the binding of native human neutrophil MPO to A. actinomycetemcomitans by an elution procedure dependent on the cationic detergent cetyltrimethylammonium bromide. Binding of native MPO was rapid and reached apparent equilibrium within 1 min. A proportion of binding under equilibrium conditions was saturable and highly avid, with a capacity of 4,500 sites per cell and a dissociation constant of 7.9 X 10(-10) M. At equal protein concentrations, more MPO III bound than MPO II, and more MPO II bound than MPO I. The high-avidity interaction was inhibitable with yeast mannan and with the serotype-defining mannan of A. actinomycetemcomitans. Binding was also partially reversible with yeast mannan. MPO bound to the high-avidity sites did not oxidize guaiacol but oxidized chloride, as detected by the chlorination of taurine. MPO bound to the high-avidity sites was incapable of killing A. actinomycetemcomitans alone in the presence of H2O2 and Cl-, but potentiated killing when sufficient additional MPO was provided. The killing of A. actinomycetemcomitans by the MPO-H2O2-Cl- system was inhibited by yeast mannan and a serotype-defining mannan of A. actinomycetemcomitans. We conclude that high-avidity binding of MPO to the surface of A. actinomycetemcomitans is a mannan-specific interaction and that MPO bound to the high-avidity sites is essential but not alone sufficient to kill A. actinomycetemcomitans.

Actinobacillus↗

Human immunodeficiency virus (HIV) antibody avidity testing to identify recent infection in newly diagnosed HIV type 1 (HIV-1)-seropositive persons infected with diverse HIV-1 subtypes.

A guanidine-based antibody avidity assay for the identification of recently acquired human immunodeficiency virus type 1 (HIV-1) infection was evaluated. The kinetics of maturation of antibody avidity were determined prospectively in 23 persons undergoing acute seroconversion followed for up to 1,075 days. Avidity indices (AI) of <or=0.75 and <or=0.80 reproducibly identified seroconversion within the previous 125 (95% confidence interval [CI], 85 to 164) and 142 (95% CI, 101 to 183) days, respectively. To validate the assay, 432 serum samples from newly diagnosed patients were tested by both the avidity assay and the detuned assay. Results were highly concordant (kappa value for agreement, 0.85). The avidity assay was subsequently used to screen 134 consecutive newly diagnosed patients, including 55/134 (41%) infected with non-B subtypes (A, C, D, G, CRF01, CRF02, CRF06, CRF13, and CRF16). In this cohort, 25/79 (32%) persons with the B subtype and 7/55 (13%) with non-B subtypes showed an AI of <or=0.75, and there were 16/25 (64%) and 3/7 (43%) persons, respectively, with a documented history of acute seroconversion illness within the predicted seroconversion interval. An AI of <or=0.75 was also observed for four patients (three with the B subtype and one with a non-B subtype) who presented with AIDS-defining conditions. In multivariate analysis, an AI of <or=0.75 was associated with younger age, higher HIV-1 plasma RNA load, and being born in the United Kingdom or Ireland rather than in Africa but not with gender, ethnicity, risk group, HIV-1 subtype, or CD4 counts. In conclusion, HIV antibody avidity testing provides a reliable method for identifying recently acquired HIV-1 infection. Results are affected by advanced disease and should therefore be interpreted in the context of other clinical parameters.

Adult↗

Application of low-avidity immunoglobulin G studies to diagnosis of Epstein-Barr virus infectious mononucleosis.

Single serum samples from 121 patients suffering from clinical infectious mononucleosis were tested by an indirect immunofluorescence assay for avidity of Epstein-Barr virus (EBV) capsid antigen immunoglobulin G (IgG), involving a wash step with phosphate-buffered saline-8 M urea. Ninety-four samples showed serological markers of recent EBV infection (presence of viral-capsid antigen-specific IgM [87 cases] and/or presence of IgG in the absence of EBV nuclear antigen antibodies [85 cases]). The remaining 27 cases had serological evidence of prior infection (presence of viral-capsid IgG and EBV nuclear antibodies and absence of IgM). In the avidity assay, 89 samples from patients with recent infection showed low-avidity IgG and 25 samples from patients with prior infection had high-avidity IgG. The avidity assay showed a sensitivity that was at least equal to those of classical serological procedures for diagnosing EBV infectious mononucleosis. Further studies are necessary, however, to establish the specificity of the assay and the duration of low-avidity antibodies.

Adolescent↗

Use of immunoglobulin G avidity assays for differentiation of primary from previous infections with West Nile virus.

Since its introduction in 1999, West Nile virus (WNV) infections have spread rapidly across the North American continent. Diagnosis of acute WNV infection by detection of WNV-specific immunoglobulin M (IgM) is complicated by the persistence of detectable IgM for more than 1 year in some patients. IgG antibody avidity testing was assessed as a supplemental assay in the diagnosis of current infections. Three groups of serum samples were assayed in parallel by two different IgG avidity test systems (indirect immunofluorescence test [IIFT] and prototype enzyme-linked immunosorbent assay [ELISA]; EUROIMMUN, Luebeck, Germany). Group I (40 sera taken between 2 and 9 days after the onset of influenza-like symptoms) and group II (40 sera taken between 10 and 43 days after onset) were acute and convalescent specimens from patients with a positive anti-WNV IgM test (ELISA; Focus Diagnostics, Cypress, CA). Group III consisted of 43 patient sera collected between 6 and 12 months after infection. IgG antibodies specific for WNV were detected in 38% (ELISA) and 50% (IIFT) of group I sera, in 90% (ELISA and IIFT) of group II sera, and in 100% (ELISA and IIFT) of group III sera. Low-avidity IgG antibodies were demonstrated in 86% (ELISA) and 95% (IIFT) of IgG-positive patient samples taken between 2 and 43 days after the onset of symptoms (groups I and II). High-avidity IgG antibodies were detected in 100% of group III sera obtained 6 months or more after the onset of symptoms (ELISA and IIFT). IgG avidity tests for WNV infections are rapid and simple to perform. The determination of IgG avidity provides additional diagnostic certainty in differentiating between recently acquired and previous infections with WNV.

Antibodies, Viral↗

Induction of higher-avidity human CTLs by vector-mediated enhanced costimulation of antigen-presenting cells.

The efficacy of antigen-specific CD8(+) CTLs depends not only on the quantity of CTLs generated but also perhaps, more importantly, on the avidity of the CTLs. To date, however, no strategy has been shown to preferentially induce higher-avidity human CTLs. In the present study, antigen-presenting cells (APC) generated from human peripheral blood mononuclear cells were infected with a recombinant avipox vector (rF-) containing the transgenes for a triad of costimulatory molecules (human B7.1, intercellular adhesion molecule-1, and LFA-3, designated as rF-TRICOM) and then used to elicit peptide-specific CTLs from autologous T cells. Compared with peptide-pulsed noninfected APCs or peptide-pulsed APCs infected with wild-type vector, peptide-pulsed APCs infected with rF-TRICOM induced not only more CTLs but also higher-avidity CTLs; this was shown by tetramer staining, tetramer dissociation, IFN-gamma production, and cytolytic assays. Peptide-pulsed rF-TRICOM-infected dendritic cells were also shown to induce CTLs with a >10-fold higher avidity than CTLs induced using CD40L-matured dendritic cells; the use of peptide-pulsed CD40L-matured dendritic cells infected with rF-TRICOM as APCs induced CTLs of even greater avidity. To our knowledge, these studies are the first to show a methodology to induce higher-avidity human CTLs and have implications for the development of more efficient vaccines for a range of human cancers.

Antibody Affinity↗

Anticytomegalovirus IgG avidity in pregnancy: a 2-year prospective study.

OBJECTIVES: To analyze the practical use of the anticytomegalovirus IgG avidity and its impact on the follow-up of pregnancy. To evaluate the performance of IgG avidity to exclude the risk of congenital infection. METHODS: 409 IgM-positive women without a documented seroconversion were prospectively followed. Data concerning the follow-up of the pregnancies were collected (amniotic fluid puncture and samples from the offspring). These observations were compared to those of 76 seroconversions during the same period. RESULTS: High avidity excluding a primary infection within the past 3 months was observed in 270 women. As the gestational age was less than 3 months for 121 women, exclusion of a primary infection was achieved in 30% of the cases. The rate of amniotic fluid puncture was influenced by the serological result: high avidity (9%), low avidity (42%) and seroconversion (65%). CONCLUSIONS: A high avidity index during the first trimester of pregnancy could reasonably be considered as a good indicator of past infection and invasive prenatal diagnosis is not necessary. Nearly 70% of the IgM-positive women could be reassured if the first serology was systematically performed before 12 weeks of gestation.

Antibodies, Viral↗

Evaluation of an in-house specific immunoglobulin G (IgG) avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection.

This article describes the standardization and evaluation of an in-house specific IgG avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection. The test was standardized with the commercial kit ETI-CYTOK G Plus (Sorin Biomedica, Italy) using 8 M urea in phosphate-buffered saline to dissociate low-avidity antibodies after the antigen-antibody interaction. The performance of the in-house assay was compared to that of the commercial automated VIDAS CMV IgG avidity test (bioM rieux, France). Forty-nine sera, 24 from patients with a recent primary HCMV infection and 25 from patients with a long-term HCMV infection and a sustained persistence of specific IgM antibodies, were tested. Similar results were obtained with the two avidity methods. All 24 sera from patients with recently acquired infection had avidity indices compatible with acute HCMV infection by the VIDAS method, whereas with the in-house method, one serum sample had an equivocal result. In the 25 sera from patients with long-term infection, identical results were obtained with the two methods, with only one serum sample having an incompatible value. These findings suggest that our in-house avidity test could be a potentially useful tool for the immunodiagnosis of HCMV infection.

Antibodies, Viral↗

Toxoplasma-IgM and IgG-avidity in single samples from areas with a high infection rate can determine the risk of mother-to-child transmission.

Anti-Toxoplasma IgG-avidity was determined in 168 serum samples from IgG- and IgM-positive pregnant women at various times during pregnancy, in order to evaluate the predictive value for risk of mother-to-child transmission in a single sample, taking the limitations of conventional serology into account. The neonatal IgM was considered the serologic marker of transmission. Fluorometric tests for IgG, IgM (immunocapture) and IgG-avidity were performed. Fifty-one of the 128 pregnant women tested gave birth in the hospital and neonatal IgM was obtained. The results showed 32 (62.75%) pregnant women having high avidity, IgM indexes between 0.6 and 2.4, and no infected newborn. Nineteen (37.25%) had low or inconclusive avidity, IgM indexes between 0.6 and 11.9, and five infected newborns and one stillbirth. In two infected newborns and the stillbirth maternal IgM indexes were low and in one infected newborn the only maternal parameter that suggested fetal risk was IgG-avidity. In the present study, IgG-avidity performed in single samples from positive IgM pregnant women helped to determine the risk of transmission at any time during pregnancy, especially when the indexes of the two tests were analysed with respect to gestational age. This model may be less expensive in developing countries where there is a high prevalence of infection than the follow-up of susceptible mothers until childbirth with monthly serology, and it creates a new perspective for the diagnosis of congenital toxoplasmosis.

Animals↗

T cell tolerance based on avidity thresholds rather than complete deletion allows maintenance of maximal repertoire diversity.

Given the flexible nature of TCR specificity, deletion or permanent disabling of all T cells with the capacity to recognize self peptides would severely limit the diversity of the repertoire and the capacity to recognize foreign Ags. To address this, we have investigated the patterns of CD8+ CTL reactivity to a naturally H-2Kb-presented self peptide derived from the elongation factor 1alpha (EF1alpha). EF1alpha occurs as two differentially expressed isoforms differing at one position of the relevant peptide. Low avidity CTLs could be raised against both variants of the EF1alpha peptide. These CTLs required 100-fold more peptide-H-2Kb complexes on the target cell compared with CTLs against a viral peptide, and did not recognize the naturally expressed levels of EF1alpha peptides. Thus, low avidity T cells specific for these self peptides escape tolerance by deletion, despite expression of both EF1alpha isoforms in dendritic cells known to mediate negative selection in the thymus. The low avidity in CTL recognition of these peptides correlated with low TCR affinity. However, self peptide-specific CTLs expressed elevated levels of CD8. Furthermore, CTLs generated against altered self peptide variants displayed intermediate avidity, indicating cross-reactivity in induction of tolerance. We interpret these data, together with results previously published by others, in an avidity pit model based on avidity thresholds for maintenance of both maximal diversity and optimal self tolerance in the CD8+ T cell repertoire.

Animals↗

Reliability of low-avidity IgG and of IgA in the diagnosis of primary infection by rubella virus with adaptation of a commercial test.

The detection of IgA and low-avidity IgG and antibodies in serum is a potentially useful marker of recent infection by a microorganism. We studied the reliability of IgG avidity and presence of IgA for the diagnosis of recent acute infection by rubella virus. Low-avidity IgG (Avy-EIA test) was determined with a modified commercial test using 8 molar urea (indirect ELISA, DiaSorin, Italy) and IgA was determined with a homemade indirect ELISA test. Twenty-five patients with recent primary infection by rubella virus (group I) and 50 healthy subjects (group II) were studied. In group I low-avidity IgG varied between 100 and 0% (67.3+/-21.8%); IgA was present in 24 patients (96%). In group II low-avidity IgG varied from 50.4 to 0% (19.8+/-16.9%). IgA was present in 2 subjects (4%). The sensitivity of the Avi-EIA and the IgA test was 92 and 96%, respectively; specificity was 100 and 96%, respectively. We conclude that both low-avidity IgG and IgA tests are helpful and reliable for the diagnosis of recent primary infection.

Adult↗

High avidity melanoma-reactive cytotoxic T lymphocytes are efficiently induced from peripheral blood lymphocytes on stimulation by peptide-pulsed melanoma cells.

To design an efficient procedure to expand high avidity melanoma reactive T cells and to perform immunotherapies, we compared conditions of peripheral blood lymphocyte (PBL) stimulation by Melan-A/MART-1 peptides. Avidity of induced CTLs was evaluated by measuring their lysis and cytokine secretion to peptide-pulsed transporter-associated protein-deficient cells and to melanoma cells. In side-by-side experiments, we show that melanoma cells, either allogeneic or autologous, induced the growth of high avidity Melan-A-reactive CTLs from all donors, whereas essentially low avidity T cells were induced by peptide-pulsed PBLs. We also show that at least two cytokines, interleukin-6 and interleukin-2, were required to promote the growth of high avidity CTLs. Once sorted by tetramer labeling or cloning, the specificity and reactivity to tumor cells of peptide-specific T cells induced by allogeneic melanoma cells were confirmed. We then describe a relatively simple and efficient procedure that allowed us to obtain systematically high amounts (in the range of billion) of high avidity Melan-A/ MART-1-specific T cells from the PBLs of HLA-A2 melanoma patients and healthy donors in 3 months. Because this antigen is expressed by most melanoma tumors, this procedure should be useful for checking the efficiency of adoptive immunotherapy of melanoma tumors and using functionally well-defined Melan-A/MART-1-specific CTLs in a large group of patients.

Amino Acid Sequence↗

[Dynamic study of anti-HIV antibody avidity after cellular immunity restoration under antiretroviral treatment].

We have studied the evolution of the avidity of anti-HIV antibodies, in 14 infected patients with Aids, including 11 patients with severe immunodeficiency at Aids stage and under active antiretroviral therapy (HAART), and 3 non-treated patients with moderate immunodeficiency. These patients have been followed up to 4 years, every 4 months the first year and every 6 months the three others, with HIV1 RNA viral load, CD4 and CD8 cells dosages and anti-HIV avidity measurements (Axsym HIV-1/2), using 1 M guanidine denaturation. A rapid decrease of the viral load was observed under Haart, inducing immune restoration with CD4 and CD8 cells increases (10 and 2-fold respectively). The decrease of anti-HIV avidity (- 20%) has been observed after 5 to 8 months under Haart, with a return to baseline value (84%). The quick restoration of CD4 cells with a persistence of viral antigens at the beginning of treatment has facilitated the selection of novel naive B lymphocytes producing low-affinity antibodies, measured by the decrease of global anti-HIV avidity. The reduction or even clearance of viral antigens under Haart could secondarily induce the selection of B lymphocytes with higher antibody affinity and therefore higher anti-HIV avidity. Thus, this avidity measurement could be used to assess the functional activity of CD4 cells restoration in HIV infected patients under Haart.

Acquired Immunodeficiency Syndrome↗

Anti-HIV-1 antibody avidity is correlated with clinical status in infected children.

Avidity of antibodies to gp41 and p24, CD4 cell count, and free and dissociated p24 antigen were assessed in 37 HIV-1-infected children, in order to evaluate whether the avidity of anti-HIV-1 antibodies is related to the disease status in perinatally infected children. The HIV-infected children were divided into two groups. Group 1 included 25 children: 2 not symptomatic (CDC category N), 12 mildly (A) and 11 moderately (B) symptomatic; group 2 included 12 children with severe clinical manifestations (C). The same parameters were assayed longitudinally in four children: two with long-term nonprogressive disease and two with fatal outcome. Antibody avidity was significantly higher in group 1 than in group 2 children (7272 +/- 4788 vs. 2624 +/- 1344 D50, p < 0.01), as was the CD4 cell count (1295 +/- 1122 vs. 348 +/- 488 cells/mm3, p < 0.01). No differences between the two groups were observed in either free or dissociated p24 antigen. Combined measures of antibody avidity and CD4 cell count showed the best correlation with the clinical status (r = 0.57, p = 0.001). In the two children with nonprogressive disease the antibody avidity remained high throughout the follow-up, whereas in those with clinical deterioration its decline preceded, by at least 8 months, the drop in CD4 cells and, by at least 23 months, the appearance of AIDS. In conclusion the avidity of anti-HIV-1 antibodies is reduced in HIV-infected children with advanced disease state and seems an earlier predictor of disease progression than CD4 cell count.

Antibody Affinity↗

[The value of the immunoglobulin G avidity test for the serologic diagnosis of brucellosis].

In order to investigate the value of immunoglobulin G (IgG) avidity test for the serological diagnosis of Brucella infections, a total of 118 patients (74 male, 44 female; mean age: 43 +/- 18.7 years) were included into the present study. The patients have been diagnosed with the characteristic clinical findings, > or = 1/160 antibody titers in standard tube agglutination (STA) test and/or blood culture positivities. Brucella spp. have been grown in blood cultures of 78 patients, and STA test results were found positive (> or = 1/160 titers) in 117 patients. The diagnosis of a patient with 1/80 STA titer was based on the blood culture positivity. By enzyme immunoassay (ELISA), 3 patients (2.5%) were found positive for IgM, 14 (11.9%) were positive for IgG, and 101 (85.6%) were positive for both IgM and IgG. The patients who were found IgG positive have been grouped according to their duration of complaints. Group 1 included 99 patients with the history of brucellosis < or = 6 months, and group 2 included 16 patients with the history of brucellosis > 6 months. IgG avidity test was performed by ELISA in 115 IgG positive serum samples, with the denaturation substance (8 M urea). The cut-off value for IgG avidity index (AI) was accepted as 40%, and the avidity maturation period was defined as 6 months. As a result, the rates of patients who had low IgG AI in group 1 and 2 were found as 91.9% and 43.7%, respectively, while these rates were 8.1% and 56.3% for the presence of high IgG AI, respectively. The rate of low AI in group 1 was an expected result, while the rate was more than expected in group 2, indicating that Brucella antibodies with low avidity indices would not be helpful for the diagnosis of a recent infection, while Brucella antibodies with high avidity indices would be useful for the elimination of a recent infection.

Adolescent↗

[Avidity of anti-glomerular basement membrane autoantibodies was associated with renal pathological damage].

OBJECTIVE: To investigate the avidity of anti-glomerular basement membrane (GBM) antibodies and their association with clinical and pathological parameters of patients with anti-GBM disease. METHODS: Sera from 52 patients and serial samples from 11 patients with anti-GBM disease, diagnosed in our hospital in the last 11 years, were collected. Purified bovine alpha chain non-collagen 1 domains of type IV collagen [alpha(IV)NC1] was employed to exam avidity of anti-GBM antibodies using antigen-inhibition enzyme-linked immunosorbent assay (ELISA). The amount of alpha(IV)NC1 needed for 50% inhibition of antibody binding was compared between patients with different clinical and pathological manifestations. RESULTS: After the sera were diluted to give the same absorbance, the amount of alpha(IV)NC1 used was different among different patients, with an average at 0.625 microg (0.02-20 microg). A significant correlation was observed between the amount of alpha(IV)NC1 used and the percentage of glomerular crescents (P = 0.001). Higher avidity of anti-GBM antibodies predicted a higher percentage of glomerular crescents (R(2) = 0.58, P = 0.000). No correlation was observed between avidity and age, gender, interval between onset and diagnosis and other clinical data. No changing avidity was observed in serial samples with time. CONCLUSION: Affinity maturation might have been completed by the time that patients presented with anti-GBM disease. The different avidity of anti-GBM antibodies was associated with the degree of renal damage and might play a key role in the pathogenesis of anti-GBM disease.

Adolescent↗

Use of IgG avidity to indirectly monitor epizootic transmission of sin nombre virus in deer mice (Peromyscus maniculatus).

An IgG avidity assay was developed to differentiate deer mice that had recently acquired Sin Nombre virus (SNV) from those that were infected in the distant past. Using this procedure, low avidity antibodies were predominantly detected in experimentally infected deer mice (89.5%) within the first 30 days post-inoculation. The assay was then applied to sera from naturally infected deer mice collected during a field investigation associated with a cluster of hantavirus pulmonary syndrome cases. A higher proportion of seropositive mice collected during the outbreak had serum with low avidity antibodies (16.7%) when compared with mice trapped four months later (5.7%). Sin Nombre virus RNA was detectable in blood in a similar fraction of low- (45%) and high- (38.7%) avidity groups. Non-adult mice were more likely to contain low-avidity antibodies (44.4%) than were adults (9.6%). Our results indicate that the IgG avidity assay shows promise as a tool to better characterize epizootic intensity and to identify factors involved in SNV transmission.

Animals↗