Synthesis and preliminary biological evaluation of a 99mTc‐labeled hypericin derivative as a necrosis avid imaging agent

Mono‐[¹²³I]iodohypericin and mono‐[¹²³I]iodohypericin monocarboxylic acid are iodine‐123‐labeled hypericin derivatives which have shown great promise in preclinical studies as necrosis avid imaging agents in animal models of infarction. In view of the more attractive properties of a ^99mTc‐labeled hypericin derivative, we have synthesized a conjugate of protohypericin monocarboxylic acid with S‐benzoylmercaptoacetyldiglycyl‐diaminopentane in an overall yield of 15%. The conjugate was labeled with technetium‐99m by exchange labeling at pH 10 in a labeling yield of 95% followed by photocyclization to yield ^99mTc‐mercaptoacetyldiglycyl‐1,5‐diaminopentylene hypericincarboxamide (^99mTc‐13). The negatively charged ^99mTc‐13 complex was purified by reversed phase high‐pressure liquid chromatography and the log P_7.4 was determined to be 2.36. In normal NMRI mice, the complex showed slow hepatobiliary clearance while plasma clearance was rapid. The tracer was evaluated in rats with reperfused hepatic infarction by ex vivo autoradiography, gamma counting and histochemical techniques. Unlike the radioiodinated hypericin derivatives, the new tracer agent did not show preferential uptake in necrotic tissue on autoradiography and gamma counting techniques. Conjugation of hypericin with a ^99mTc‐chelate, resulting in a change in size, charge and lipophilicity, had a profound effect on the necrosis avidity of the tracer agent. The results show that ^99mTc‐13 is not suitable for imaging necrosis. Copyright © 2008 John Wiley & Sons, Ltd.     

Evaluating the population measles susceptibility in Tianjin,China

BackgroundMeasles is a highly infectious illness requiring herd immunity of 95% to interrupt transmission. China has not reached elimination goals despite high vaccination coverage. We estimated the population susceptibility against measles in Tianjin, China and to tailor awareness raising activities in the measles elimination plan.MethodsAge-specific measles seroprevalence was evaluated by Enzyme-Linked Immunosorbent Assay (ELISA) on 12,164 individual aged 0–44 years in 2009–2018. Measles IgG avidity testing was performed to confirm the relationship of the waning immunity after vaccination and secondary vaccination failures (SVF) on 324 confirmed measles cases in 2013–2018.Results11,108 samples (91.32%) tested positive for measles IgG, 239 (1.96%) tested as equivocal and 817 (6.72%) were negative. The age distribution of measles cases in Tianjin followed a U-shaped curve and was highest for those at <8 months and again at 20–39 years which correlated closely with the age distribution of measles susceptibility based on measles IgG antibody status (r = 0.72, P < 0.001). The seropositivity rate and antibody geometric mean concentration (GMC) for the 2018 study population were significantly lower (χ² = 7.45, P = 0.006 and t = 12.01, P < 0.001) compared to 2009. The multivariate stepwise logistic regression analysis showed that age and region were the risk factors for both measles seropositivity rate and GMC after vaccination. The proportion of high avidity cases increased with age, being significantly higher in 75.31% of cases in patients aged 30–34 years (χ² = 18.04, P = 0.003).ConclusionsHigh immunization coverage in children alone will not be adequate to realizing sufficient levels of population herd immunity, particularly given that the potential susceptibility window in adult. Implementation of supplemental immunization activity (SIA) targeted to appropriate group aged 30–34 years is recommended.     

Serum IgG titres,but not avidity,correlates with neutralizing antibody response after H5N1 vaccination

Background

Influenza H5N1 virus constitutes a pandemic threat and development of effective H5N1 vaccines is a global priority. Anti-influenza antibodies directed towards the haemagglutinin (HA) define a correlate of protection. Both antibody concentration and avidity may be important for virus neutralization and resolving influenza disease.

Methods

We conducted a phase I clinical trial of a virosomal H5N1 vaccine adjuvanted with the immunostimulating complex Matrix M™. Sixty adults were intramuscularly immunized with two vaccine doses (21 days apart) of 30 μg HA alone or 1.5, 7.5 or 30 μg HA adjuvanted with Matrix M™. Serum H5 HA1-specific antibodies and virus neutralization were determined at days 0, 21, 42, 180 and 360 and long-term memory B cells at day 360 post-vaccination. The binding of the HA specific antibodies was measured by avidity NaSCN-elution ELISA and surface plasmon resonance (SPR).

Results

The H5 HA1-specific IgG response peaked after the second dose (day 42), was dominated by IgG1 and IgG3 and was highest in the adjuvanted vaccine groups. IgG titres correlated significantly with virus neutralization at all time points (Spearman r ≥ 0.66, p < 0.0001). By elution ELISA, serum antibody avidity was highest at days 180 and 360 post vaccination and did not correlate with virus neutralization. Long-lasting H5 HA1-specific memory B cells produced high IgG antibody avidity similar to serum IgG.

Conclusions

Maturation of serum antibody avidity continued up to day 360 after influenza H5N1 vaccination. Virus neutralization correlated with serum H5 HA1-specific IgG antibody concentrations and not antibody avidity.     

Human Antibody Responses to Avian Influenza A(H7N9) Virus, 2013

Understanding host antibody response is crucial for predicting disease severity and for vaccine development. We investigated antibody responses against influenza A(H7N9) virus in 48 serum samples from 21 patients, including paired samples from 15 patients. IgG against subtype H7 and neutralizing antibodies (NAbs) were not detected in acute-phase samples, but ELISA geometric mean titers increased in convalescent-phase samples; NAb titers were 20–80 (geometric mean titer 40). Avidity to IgG against subtype H7 was significantly lower than that against H1 and H3. IgG against H3 was boosted after infection with influenza A(H7N9) virus, and its level in acute-phase samples correlated with that against H7 in convalescent-phase samples. A correlation was also found between hemagglutinin inhibition and NAb titers and between hemagglutinin inhibition and IgG titers against H7. Because of the relatively weak protective antibody response to influenza A(H7N9), multiple vaccinations might be needed to achieve protective immunity.     

Age-related differences in antibody avidities to pertussis toxin and filamentous hemagglutinin in a healthy Japanese population

To gain insights into the current Japanese pertussis immunization schedule, we examined the distributions of antibody titers and avidities to pertussis toxin (PT) and filamentous hemagglutinin (FHA) in 460 Japanese healthy subjects (aged 1–60?years) based on age category. Our avidity enzyme-linked immunosorbent assays revealed that young children aged 1–2?years, which corresponded to ages after receiving primary and/or booster pertussis vaccinations, had relatively high-avidity anti-PT IgG (mean avidity index [AI], 40.5%) compared with other age groups (AI, 26.5–31.9%); however, they had relatively low-avidity anti-FHA IgG (AI, 41.8%). In contrast, children aged 3–6?years had both low-avidity anti-PT IgG (AI, 26.5%) and low-avidity anti-FHA IgG (AI, 40.4%). A significant age-related difference in anti-PT IgG avidity was observed between children aged 1–2?years and 3–6?years (P?<?0.05); however, the difference in anti-FHA IgG avidity was not significant. The anti-PT IgG avidity was positively correlated with the antibody titer, especially among children aged 1–15?years (r_s?=?0.508–0.685; P?<?0.01), indicating that the avidity of vaccine-induced anti-PT IgG decreases with decreasing IgG antibody titer to PT. Our findings strongly suggest that vaccine-induced anti-PT IgG avidity rapidly wanes after vaccination, but this is not observed for anti-FHA IgG avidity. Because children aged 3–6?years have both low-quantity and low-quality antibodies against PT, an additional booster vaccination with acellular pertussis vaccines is required for such children in Japan.     

CTL quality and the control of human retroviral infections

The CTL response plays a central part in deciding the outcome of viral infections. Evidence from host and viral genetics, gene expression microarrays and assays of T‐cell phenotype and function indicate that individual differences in the efficiency of the virus‐specific CTL response strongly determine the outcome of infection with the human retroviruses HTLV‐1 and HIV‐1. It is now believed that differences in anti‐viral CTL efficiency or “quality” at the single‐cell level are critical in determining the efficacy of the host response to viruses. However, it is difficult to identify and quantify the reasons for this apparent individual variation in CTL efficiency, because of the chronic course of infection and the dynamical complexity of the equilibrium that is established between the virus and the host immune response. Specifically, it is unclear whether the observed variations among infected hosts, i.e. in the frequency, phenotype and function or quality of T cells, are the causes or effects – or both – of the variation in the efficiency of virus control.     

Separation and isolation of lymphocyte subpopulations of different affinity for the antigen

In order to separate, isolate, and determine the number and distribution of the subpopulations of lymphocytes of diverse affinities that are present in an immune response toward a single hapten, anti-trinitrophenyl (TNP) lymphocytes from immunized animals were purified by cell chromatography. Non-adherent spleen cells were passed through a column consisting of TNP-substituted polyacrylamide beads. The retained cells were eluted by applying a linear concentration gradient of TNP-lysine. Elution profiles having a limited number of peaks were obtained in all cases. The avidity of the cells in each fraction was measured by inhibition of formation of immune rosettes by free hapten. Results showed that each peak was located along the gradient according to its affinity since there was a direct correlation between the affinity and the concentration of hapten needed for the elution. The cells in each peak appeared to belong to a homogeneous subpopulation as shown by the slope of the curves obtained in the determination of avidity, suggesting that each peak corresponded to one expanded clone.     

Role of the CDR1 region of the TCR {beta} chain in the binding to purified MHC-peptide complex

Single alanine substitutions were introduced into the CDR1 regionof the ß chain of a K^d-restricted TCR. Mutants andwild-type TCR were attached to the chain of the CD3 complexand expressed at the surface of a rat basophil cell line. Transfectantswere tested for the binding of purified soluble K^d-peptlde complexes.With this experimental system, accessory molecules are unlikelyto play a major role and the contribution of each residue tothe interaction can be addressed. Results show that all positionsin the CDR1 region are involved in the binding to the K^d-peptidecomplex but at varying degrees. These effects are discussedin relation to a molecular model of the TCR. Comparison of theseresults with previous data obtained in a T cell hybridoma systemsuggests the existence of a threshold in the TCR affinity necessaryfor mature T cell activation.     

A new human IgG avidity test,using mixtures of recombinant antigens (rROP1, rSAG2, rGRA6), for the diagnosis of difficult-to-identify phases of toxoplasmosis

The preliminary diagnostic utility of two mixtures of Toxoplasma gondii recombinant antigens (rROP1+rSAG2 and rROP1+rGRA6) in IgG ELISA and IgG avidity test has been evaluated. A total of 173 serum samples from patients with toxoplasmosis and seronegative people were examined. The sensitivity of IgG ELISA for rROP1+rSAG2 and rROP1+rGRA6 was 91.1% and 76.7%, respectively, while the reactivity for sera from patients where acute toxoplasmosis was suspected was higher, at 100% and 95.4%, respectively, than for people with chronic infection, at 88.2% and 70.6%. In this study a different trend in avidity maturation of IgG antibodies for two mixtures of proteins in comparison with native antigen was observed. The results suggest that a new IgG avidity test using the mixtures of recombinant antigens may be useful for the diagnosis of difficult-to-identify phases of toxoplasmosis. For this reason, selected mixtures after the additional tests on groups of sera with well-defined dates of infection could be used as a better alternative to the native antigens of the parasite in the serodiagnosis of human T. gondii infection.     

Murine monoclonal antibodies to DNA. A comparison of MRL/lpr NZB/W and chronically graft-versus-host-diseased mice

Hybridomas producing monoclonal antibodies to DNA were prepared from NZB/W F1 (n = 20), MRL/lpr (n = 13), mice with a chronical graft versus-host-disease (GVHD) (n = 8) and polyclonally stimulated mice (n = 9). Screening was performed by means of an anti-DNA ELISA. Reaction patterns in four different anti-DNA assays (anti-DNA ELISA, indirect immunofluorescence on Crithidia luciliae, PEG assay and Farr assay) as well as avidity and cross-reactivity of these monoclonals were studied in relation to anti-DNA (sub)class and murine origin of the clones. It was found that monoclonal anti-DNA derived from mice with chronic GVHD did not differ from monoclonal anti-DNA derived from NZB/W F1 or MRL/lpr mice, with respect to isotype distribution, avidity towards DNA, cross-reactivity and assay behaviour in the anti-DNA assays mentioned before. In contrast, monoclonal anti-DNA obtained from polyclonally stimulated mice were all of the IgM isotype and displayed a stronger cross-reactive behaviour than the other three models. Altogether, these results exclude the possibility that anti-DNA in the GVHD mice originates from the non-specific pool of natural autoantibodies and further emphasize the relevance of chronic GVHD as a murine model of systemic lupus erythematosus.