Memory immune response generated in Cercopithecus aethiops against meningococcal polysaccharide serogroup C conjugate vaccine

The chemical conjugation of bacterial polysaccharide to carrier proteins has proved to be an efficient tool to improve the immunological response against these bacterial antigens. In this study, we characterized the antibody response generated in a non-human primate model against the meningococcal capsular polysaccharide serogroup C (CCPS) conjugated to the P64k protein. Similar to licensed vaccines the CCPS conjugate is able to generate a good memory immune response with antibody titers threefold higher than the free CCPS. Three different ELISA protocols were used to measure the antibody response and the importance of the coating antigen was demonstrated. The ELISA using the derivatized CCPS showed the best results and had a high correlation with the bactericidal activity. The antibodies elicited showed a high protective capacity when assayed in the infant rat protection model.     

A novel monoclonal antibody to Neisseria meningitidis serogroup X capsular polysaccharide and its potential use in quantitation of meningococcal vaccines

A novel murine hybridoma monoclonal antibody (MAb) was produced against the capsular polysaccharide (CP) of Neisseria meningitidis serogroup X (MenX) in order to develop a sandwich enzyme linked immunosorbent assay (ELISA) for the quantitation of the meningococcal polysaccharide. The MAb only reacted with the CP from MenX and did not react with CPs from N. meningitidis serogroups A, C, Y and W (MenA, MenC, MenY, MenW). The affinity constant (Ka) of the MAb measured by non-competitive ELISA was 7.25 × 10⁷ M⁻¹. The application of this MAb in a sandwich ELISA was demonstrated by its ability to properly quantitate three lots of an experimental meningococcal CP-based vaccine. The MAb obtained in this work could be a valuable reagent for the detection and quantitation of future meningococcal vaccines containing MenX CP.     

Development and validation of ELISA for detection of antibodies to Legionella pneumophila serogroup 1, 3 and 6 in human sera

The aim of this study was to determine whether separate measurement of immunoglobulin (Ig) M and G antibodies to Legionella (L.) pneumophila serogroups (sg) 1, 3 and 6 as single antigens can facilitate an early diagnosis of Legionnaires' disease. The developed ELISA was evaluated and compared with an in-house indirect Legionella immunofluorescence antibody test (IFAT) measuring Total Ig. A total of 193 sera from 128 patients with confirmed L. pneumophila infections were used to assess the sensitivity of the developed ELISA. The sensitivity was assessed in different time-periods after onset of symptoms. It was found that the sensitivity of the test increased during the first month of infection, IgM being the most sensitive; ranging from 13% in the first week after onset of symptoms, 45% in the second week to 84% in the third week; in the fourth (and beyond) week a drop to 67% was observed. The IFAT detecting L. pneumophila sg 1-6 had a sensitivity of 11%, 27%, 80% and 59%, respectively, during these time-periods. The test with the lowest sensitivity was the IgG ELISA (0%, 21%, 36% and 52%), but by combining the IgG results with the IgM results, the overall sensitivity of the assay was improved (13%, 48%, 88% and 70%).This study confirms that detection of IgG and IgM antibodies by ELISA is an important diagnostic tool especially during the initial phase of the disease, when supported by other tests like the urinary antigen test, PCR or culture.Furthermore, we showed that the ELISA is suitable for the detection of significant changes in antibody levels in paired serum samples. It was found that the sensitivity was higher for the ELISA assays than for the IFAT. Both the in-house IgM ELISA and the IFAT had a low false positive rate, while a 14% false positive rate was found for the IgG ELISA among serum samples from patients with other infections.     

Detection of elevated antibody against calreticulin by ELISA in aged cynomolgus monkey plasma

Calreticulin (Crt) is a molecular chaperone ubiquitously present in the endoplasmic reticulum. In non-human primates, age-related occurrence of anti-Crt antibody has not been reported. We developed an ELISA assay for an anti-Crt antibody and determined the age-related increase in the levels of anti-Crt antibody in three groups of cynomolgus monkeys: juvenile (1.5 yr), young adults (5-10 yr) and aged adults (20-34 yr). Mean ± SD auto-antibody levels at 450 nm in juvenile, young adults and aged groups were 0.23 ± 0.18, 0.30 ± 0.28, and 0.55 ± 0.33, respectively. Statistically significant differences were noted in the autoantibody levels to Crt among the aged group and juvenile or young adults. This is the first report to demonstrate the expression of anti-Crt autoantibody in aged monkeys and indicates that cynomologous monkeys may serve as an appropriate nonhuman primate model for studies of age-related alteration of immune function in elderly humans. Though preliminary, this finding merits further investigation to determine the relationship between immunosenescence and expression of antibodies to Crt.     

2019年河南省健康人群流行性脑脊髓膜炎A群、C群抗体水平监测

目的了解2019年河南省健康人群流行性脑脊髓膜炎(epidemic cerebrospinal meningitis,简称流脑)A群、C群抗体水平状况,为科学制定防控策略提供依据。方法采用分层随机抽样的方法采集健康人群血清,用间接ELISA检测流脑A群、C群抗体浓度。结果共检测1 689份健康人群血清标本,其中流脑A群抗体阳性率为54.23%,抗体质量浓度中位数为2.18μg/mL;流脑C群抗体阳性率为51.51%,抗体质量浓度中位数为2.07μg/mL。不同地区之间流脑A群、C群抗体水平的差异、阳性率的差异均有统计学意义(P<0.05)。不同年龄组之间流脑A群、C群抗体水平的差异、阳性率的差异均有统计学意义(P<0.05)。结论河南省健康人群流脑A群、C群抗体水平和阳性率都比较低,有发生流脑暴发和流行的血清学基础,应加强流脑监测和预防接种的管理工作。     

B cell memory to a serogroup C meningococcal conjugate vaccine in childhood and response to booster: little association with serum IgG antibody

The maintenance of adequate serum Ab levels following immunization has been identified as the most important mechanism for individual long-term protection against rapidly invading encapsulated bacteria. The mechanisms for maintaining adequate serum Ab levels and the relationship between Ag-specific memory B cells and Ab at steady state are poorly understood. We measured the frequency of circulating serogroup C meningococcal (MenC)-specific memory B cells in 250 healthy 6- to 12-y-old children 6 y following MenC conjugate vaccine priming, before a booster of a combined Haemophilus influenzae type b-MenC conjugate vaccine and then 1 wk, 1 mo, and 1 y after the booster. We investigated the relationship between circulating MenC-specific memory B cell frequencies and Ab at baseline and following the booster vaccine. We found very low frequencies of circulating MenC-specific memory B cells at steady state in primary school-aged children and little association with MenC IgG Ab levels. Following vaccination, there were robust memory B cell booster responses that, unlike Ab levels, were not dependent on age at priming with MenC. Measurement of B cell memory in peripheral blood does not predict steady state Ab levels nor the capacity to respond to a booster dose of MenC Ag.     

Profiles of human serum antibody responses elicited by three leading HIV vaccines focusing on the induction of Env-specific antibodies

In the current report, we compared the specificities of antibody responses in sera from volunteers enrolled in three US NIH-supported HIV vaccine trials using different immunization regimens. HIV-1 Env-specific binding antibody, neutralizing antibody, antibody-dependent cell-mediated cytotoxicity (ADCC), and profiles of antibody specificity were analyzed for human immune sera collected from vaccinees enrolled in the NIH HIV Vaccine Trial Network (HVTN) Study #041 (recombinant protein alone), HVTN Study #203 (poxviral vector prime-protein boost), and the DP6-001 study (DNA prime-protein boost). Vaccinees from HVTN Study #041 had the highest neutralizing antibody activities against the sensitive virus along with the highest binding antibody responses, particularly those directed toward the V3 loop. DP6-001 sera showed a higher frequency of positive neutralizing antibody activities against more resistant viral isolate with a significantly higher CD4 binding site (CD4bs) antibody response compared to both HVTN studies #041 and #203. No differences were found in CD4-induced (CD4i) antibody responses, ADCC activity, or complement activation by Env-specific antibody among these sera. Given recent renewed interest in realizing the importance of antibody responses for next generation HIV vaccine development, different antibody profiles shown in the current report, based on the analysis of a wide range of antibody parameters, provide critical biomarker information for the selection of HIV vaccines for more advanced human studies and, in particular, those that can elicit antibodies targeting conformational-sensitive and functionally conserved epitopes.     

Relation of human serum antibody against Staphylococcus epidermidis cell surface polysaccharide detected by enzyme-linked immunosorbent assay to passive protection in the mouse

A specific and rapid enzyme-linked immunosorbent assay (ELISA) has been applied for the detection of immunoglobulins to Staphylococcus epidermidis cell surface polysaccharides in human serum. Positive IgG, IgM and IgA titres of more than 1: 6400, 1: 1600 and 1: 400 were observed with this assay against passive protective human serum. However, IgG, IgM and IgA titres of less than 1: 400, 1: 100 and 1: 50 were shown in non-protective serum. When the cross-reactivity of passive protective human serum to homologous and heterologous cell surface polysaccharides was examined by inhibition test with ELISA, remarkable inhibition was shown with homologous cell surface polysaccharide, whereas no inhibition was observed with heterologous substances. According to these results, the quantitation of human serum antibody by the ELISA method against Staph. epidermidis cell surface polysaccharide was found to be significant for the demonstration of passive protective activities against Staph. epidermidis.     

Relation of human serum antibody against Staphylococcus epidermidis cell surface polysaccharide detected by enzyme-linked immunosorbent assay to passive protection in the mouse

A specific and rapid enzyme-linked immunosorbent assay (ELISA) has been applied for the detection of immunoglobulins to Staphylococcus epidermidis cell surface polysaccharides in human serum. Positive IgG, IgM and IgA titres of more than 1:6400, 1:1600 and 1:400 were observed with this assay against passive protective human serum. However, IgG, IgM and IgA titres of less than 1:400, 1:100 and 1:50 were shown in non-protective serum. When the cross-reactivity of passive protective human serum to homologous and heterologous cell surface polysaccharides was examined by inhibition test with ELISA, remarkable inhibition was shown with homologous cell surface polysaccharide, whereas no inhibition was observed with heterologous substances. According to these results, the quantitation of human serum antibody by the ELISA method against Staph. epidemidis cell surface polysaccharide was found to be significant for the demonstration of passive protective activities against Staph. epidermidis.     

Modelling cost effectiveness of meningococcal serogroup C conjugate vaccination campaign in England and Wales

ObjectivesTo assess the cost effectiveness of a meningococcal serogroup C conjugate vaccination campaign in 0-17 year olds.DesignCost effectiveness analysis from the perspective of the healthcare provider.SettingEngland and Wales.ResultsIn 1998-9, immediately before the introduction of meningococcal C vaccination, the burden of serogroup C disease was considerable, with an estimated 1137 cases in people aged 0-17 years and at least 72 deaths. The vaccination campaign is estimated to have cost between £126m ($180m, €207m) and £241m ($343m, €395m), depending on the price of the vaccine. Under base case assumptions the cost per life year saved from the vaccination campaign is estimated to be £6259. School based vaccination was more cost effective than general practice based vaccination because of lower delivery costs. Immunisation of infants aged under 1 year was the least cost effective component of the campaign because, although this maximises the life years gained, the three dose schedule required is more expensive than other methods of delivery. Estimates of the cost per life year saved were sensitive to assumptions on the future incidence of disease and the case fatality ratio.ConclusionsMeningococcal C vaccination is likely to be more cost effective in all age groups when the incidence of disease is high. It is also more cost effective when given to children aged 1-4 (by general practitioners) and to children and young people aged 5-17 years at school than when administered to infants under 12 months of age or young people aged 16-17 years who are not at school.

What is already known on this topic

The burden of group C meningococcal disease in England and Wales in the late 1990s was considerableIn November 1999 the United Kingdom was the first country to introduce mass vaccination against group C meningococcal diseaseThere are no published economic evaluations of the vaccination campaign

What this study adds

This economic evaluation supports the introduction of the meningococcal C vaccineSchool based vaccination is more cost effective than routine vaccination of infants because delivery costs are lower and fewer doses are required     

C群奈瑟氏脑膜炎球菌荚膜多糖-TT结合疫苗的制备及其在小鼠体内免疫原性的研究

将C群脑膜炎球菌荚膜多糖以ADH作为间隔剂与TT结合形成GCMP-TT结合疫苗,然后用此结合疫苗免疫NIH小鼠,结果显示使用GCMP免疫小鼠后仅能产生较低水平抗GCMP的IgG抗体,而用GCMP-TT免疫后小鼠血清中产生了较GCMP免疫显著增高抗GCMP的IgG抗体,并且GCMP-TT组第二次和第三次免疫后与初次免疫相比,IgG抗体水平均有显著升高(P<0.01),表明GCMP-TT结合疫苗具有免疫记忆和再次免疫加强应答效应。补体介导的血清抗体体外杀菌试验结果证明,GCMP-TT结合疫苗组免疫小鼠诱导的抗体IgG比GCMP组具有增强的体外杀菌活性。     

Demonstration of cross-reactive antibodies to mycoplasmas in human sera by ELISA and immunoblotting

Varying levels of cross-reactivity to some mycoplasma species were observed in the sera of patients infected with Mycoplasma pneumoniae and even in normal human sera by enzyme-linked immunosorbent assay (ELISA). The absorption of the patients' sera with M. pneumoniae lysate showed the decrease in ELISA titers not only to M. pneumoniae, but also to other mycoplasma species. These results suggested the existence of cross-reactive antibodies to mycoplasmas in human sera. Cross-reactive antibodies to M. pneumoniae and other mycoplasmas in the patients' sera were also demonstrated by Western blotting technique.     

Kinetic, affinity, and diversity limits of human polyclonal antibody responses against tetanus toxoid

Due to technical limitations, little knowledge exists on the composition of Ag-specific polyclonal Ab responses. Hence, we here present a molecular analysis of two representative human Ab repertoires isolated by using a novel single-cell cloning approach. The observed genetic diversity among tetanus toxoid-specific plasma cells indicate that human polyclonal repertoires are limited to the order of 100 B cell clones and hypermutated variants thereof. Affinity and kinetic binding constants are log-normally distributed, and median values are close to the proposed affinity ceilings for positive selection. Abs varied a million-fold in affinity but were restricted in their off-rates with an upper limit of 2 x 10(-3) s(-1). Identification of Abs of high affinity without hypermutations in combination with a modest effect of hypermutations on observed affinity increases indicate that Abs selected from the naive repertoire are not only of low affinity but cover a relatively large span in affinity, reaching into the subnanomolar range.     

Competitive inhibition by human sera of mouse monoclonal antibody binding to glycoproteins C and D of herpes simplex virus types 1 and 2.

A competitive enzyme-linked immunosorbent assay was used to test for human antibodies to antigenic sites on herpes simplex virus (HSV) glycoproteins C and D, which are recognized by mouse monoclonal antibodies. Antibodies capable of blocking the monoclonal antibodies were detected in the human sera, and the inhibition of binding correlated with the histories of herpetic infections. The binding of monoclonal antibody to glycoprotein C of HSV type 2 was inhibited primarily by sera from patients with recurrent herpes genitalis; however, the binding of the monoclonal antibodies to gC of HSV type 1 was inhibited by sera from patients previously infected with either HSV type 1 or HSV type 2. The observations suggest that the antigenic sites defined by the mouse monoclonal antibodies are recognized by the human host.     

Immune responses to hapten--lipopolysaccharide conjugates. 1. Modulation of in vivo antibody responses to the polysaccharide

The immune responses of mice to various lipopolysaccharides (LPS) and hapten-LPS conjugates were compared. We found that some strains of mice ($\text{A}\text{J}$ and BALB/c) produced equivalent amounts of anti-LPS antibody after the injection of either LPS or hapten-LPS conjugates. In contrast, however, other strains of mice (C57BL/6J, C3H/St, DBA/1J, DBA/2J, and Swiss) produced fewer anti-LPS-antibody-secreting cells after stimulation with hapten-LPS conjugates than did mice injected with unsubstituted LPS. The covalent coupling of hapten to LPS changed neither the mitogenic capacity nor the antigenicity of the LPS. The differences in the magnitude of antibody responses to hapten-LPS and LPS in these latter strains of mice occurred in the absence of mature T lymphocytes and was restricted to the primary immune response. Furthermore, these differential responder mice (C57BL/6J) did produce anti-LPS antibody when primed with LPS before challenge with the hapten-LPS conjugate. These data are discussed with respect to both the modulatory capacity of the hapten-LPS in the regulation of the primary immune response to LPS and the biochemical and structural requirements of the hapten-LPS conjugate for immunogenicity.     

hGH单克隆抗体的筛选及双抗体夹心ELISA检测方法的建立

采用杂交瘤法制备单克隆抗体,并用辛酸-硫酸铵法纯化单抗,通过ELISA方法和Western blotting测定抗体的效价与特异性,并进行抗体类型、相对亲和力测定;应用纯化的单抗建立hGH双抗体夹心ELISA检测方法。筛选出两株可以稳定分泌抗hGH单抗的杂交瘤细胞株,分别命名为3E11、2G9,抗体类型均为IgG1,抗体滴度均可达10-10,特异性好,相对亲和力高,以筛选到的两株单抗建立的双抗夹心ELISA法线性范围为0.09～1.5625ng/mL,R2>0.9,灵敏度为0.09ng/mL。筛选出高效抗hGH的单抗,并建立了hGH双抗体夹心ELISA检测方法。