A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology

Marek’s disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek’s disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant—GX0101Δpp38—and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas—namely, 4F9, 31G7, 34F2, and 35G9—were demonstrated to secrete specific antibodies against MDV-1’s pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography–tandem mass spectrometry (LC–MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses—especially for large DNA viruses such as herpesviruses.     

Cat scratch disease: analysis of 130 seropositive cases

 To clarify the clinical manifestations of cat scratch disease (CSD), we evaluated a total of 130 seropositive patients with CSD. The patients' ages ranged from 1 to 68 years; 103 (79.2%) were under 18 years of age. CSD occurred predominantly in the fall and winter months. Regional lymphadenopathy was noted in 110 (84.6%) of the cases, and the most common sites were the neck (33%), axillary (27%), and inguinal (18%) regions. One hundred of the patients (77%) had general symptoms, such as fever, headache, and malaise. The clinical manifestations of CSD showed a wide spectrum from typical or classical CSD, with regional lymphadenopathy, to atypical or systemic CSD. Of the 130 cases, 103 (79.2%) were typical CSD and 27 (20.8%) were atypical CSD. Atypical cases of CSD were commonly reported as fever of unknown origin (37.0%), neuroretinitis (22.2%), encehalopathy (14.8%), hepatosplenic granuloma (11.1%), and Parinaud's oculoglandular syndrome (7.4%). Fever of unknown origin or prolonged fever lasting more than 14 days was evident in 27 (20.8%) of the 130 cases in this study. Eleven of the 27 cases lacked lymphadenopathy. Our findings suggest that CSD is not a rare disease in Japan. The indirect fluorescent antibody (IFA) test to detect Bartonella species may provide a prompt diagnosis of CSD and facilitate appropriate therapy. Received: March 6, 2002 / Accepted: July 8, 2002     

Transfusion-associated transmission of babesiosis in New York State

BACKGROUND: Babesiosis can be life-threatening in immunocompromised individuals. Although the disease is usually transmitted by tick bite, more than 20 cases have been reported of infection transmitted by transfusion of blood or blood components obtained from apparently healthy donors from endemic areas in the United States. This report describes several recent cases of transfusion-transmitted babesiosis in New York State. STUDY DESIGN AND METHODS: Transfusion-associated incidents of babesiosis infection were identified and investigated. Seroprevalence of babesiosis in healthy blood donors in a highly endemic area was ascertained. RESULTS: In three incidents, babesiosis was diagnosed in five of eight patients given infected blood: two premature infants, an elderly patient with gastrointestinal bleeding, and two patients with thalassemia. Seroprevalence in blood donors on Shelter Island (Suffolk County, eastern Long Island), a highly endemic area, was 4.3 percent in May 1998. CONCLUSIONS: Infected donors lived in endemic areas and were asymptomatic with no history of tick bite. Blood collected in January 1997 from one donor was infectious. Those transfusion recipients who were infected were neonatal, elderly, or chronically transfused patients. Babesiosis should be included in the differential diagnosis of febrile illness in immunocompromised recipients of blood transfusion, particularly in the Northeastern United States.     

济南地区肺炎病人中军团杆菌感染状况研究

本文应用病例-对照研究方法,以IFA试验对济南铁路医院98对肺炎病例及非肺科病例Lp的感染状况作了调查。观察组抗体滴度达诊断标准者25例,总阳性率25.51%,表明肺炎病人中军团杆菌肺炎占有相当比例。感染型别主要为VI型。50~59岁年龄组阳性率最高。     

Fire ant venom hypersensitivity. I. Comparison of fire ant venom and whole body extract in the diagnosis of fire ant allergy

Levels of IgE antibody to fire ant whole body extract (FA-WBE) and fire ant venom (FAV) were evaluated in 60 patients with clinical histories of fire ant sting hypersensitivity and 11 frequently stung individuals without significant clinical reactivity to fire ant. An overlap of levels of IgE antibody to FA-WBE and FAV was seen between groups of patients with increasingly severe clinical reactivity; however, the group mean IgE antibody level of patients without significant clinical reactivity differed markedly from the group means of patients with more severe clinical reactivity (p less than 0.001). In 31 patients a good correlation (r = 0.6935) was found comparing FA-WBE IgE antibody level with FA-WBE skin tests, and in 26 patients a good correlation (r = 0.8564) was obtained comparing FAV IgE antibody levels with FAV skin tests. An excellent Spearman rank correlation coefficient (r = 0.9197) was found on comparison of FAV and FA-WBE IgE antibody levels in 71 individuals. Cross-reactivity of FAV and FA-WBE was demonstrated by the ability of these reagents to competitively inhibit the binding of IgE antibody to solid-phase FA-WBE and FAV in the radioallergosorbent test (RAST). From these results we conclude that FAV and FA-WBE both contain relevant allergens important in fire ant-allergic individuals and that skin tests and RAST are both valid as diagnostic tests for fire ant allergy. Finally, there is a good correlation between RAST and skin tests using FA-WBE or FAV as test reagents, since these reagents are highly cross-reactive as demonstrated by their ability to inhibit each other in the RAST.     

Reducing the risk of transfusion-transmitted rickettsial disease by WBC filtration, using Orientia tsutsugamushiin a model system

BACKGROUND: Careful donor screening and infectious disease marker testing have significantly reduced the incidence of transfusion-transmitted diseases and improved the safety of the blood supply. However, transfusion-transmitted diseases resulting from the use of asymptomatic yet infectious donors continue to put patients at risk. This study was undertaken to determine if third-generation WBC filters could remove Orientia tsutsugamushi-infected cells from contaminated blood. STUDY DESIGN AND METHODS: Packed RBCs were inoculated with human MNCs infected with O. tsutsugamushi at levels estimated to occur in asymptomatic infectious donors. WBC reduction was accomplished with a third-generation WBC filter. Prefiltration and postfiltration specimens were collected, serially diluted, and injected into mice to determine the infectivity of the samples. RESULTS: Mice receiving WBC-reduced packed RBCs showed no signs of illness or markers of infectivity, which suggested that a reduction of as much as 10(5) infectious rickettsiae could be achieved by filtration. CONCLUSION: The high-efficiency, third-generation, WBC-reduction filters that were tested may provide protection against the transfusion transmission of scrub typhus rickettsiae by removing from contaminated blood cells that contain intracellular bacteria.     

Improving the immunogenicity and protective efficacy of the EtMIC2 protein against Eimeria tenella infection through random mutagenesis

In recent years, directed evolution has emerged as an efficient tool to develop and identify novel protein variants. Eimeria tenella microneme-2 (EtMIC2) is a promising vaccine candidate for use against E. tenella infection; however, it only yields partial protection. The present study aimed to improve the immunogenicity and protective efficacy of EtMIC2 through random mutagenesis. Mutagenesis gene libraries of EtMIC2 were generated using error-prone polymerase chain reaction (epPCR), and the corresponding variant proteins were displayed on the yeast cell surface. Variant EtMIC2 proteins with high immunogenicity were screened through fluorescence-activated cell sorting (FACS) based on the affinity between polyclonal antibodies and antigens. Seven effective variant proteins were screened out and heterogeneously expressed in Escherichia coli as subunit vaccines. The protective efficacy of the variant proteins against E. tenella infections was then evaluated in chicken. Two variant proteins (1130 and 2119) displayed higher immunogenicity and protective efficacy than the wild-type EtMIC2 protein against E. tenella infections, increasing body weight gains and significantly decreasing lesion scores and fecal oocyst shedding, and increasing sIgA antibody production and lymphocyte proliferation. These variants displayed potential for use in the development of subunit vaccines for coccidiosis in chickens. The present results also indicate that directed evolution technology is useful for improving the immunogenicity and protective efficacy of parasite antigens.     

Role of goblet cells and mucin layer in protecting maternal IgA in precocious birds

Immune protection of the gut in early life depends on provision of maternal antibodies, particularly that of IgA. In precocial birds (in this study Gallus gallus domesticus) the egg provides the only source of maternal antibodies, IgA inclusive. The gut-life of IgA in hatchlings is expected to be brief due to antigen binding and intestinal washout, and maternal IgA is likely to be depleted prior to immune independence at 7–10 days of age in the domestic fowl. We followed the track of maternal IgA in mucosal surfaces of the fowl and describe for the first time a mechanism that might provide the means to extend the active period of maternal IgA in the gut. Maternal IgA was located in the gut, lung, and cloacal bursa in embryos and hatchlings prior to the appearance of endogenic IgA positive plasma cells (D3 in the bursa or D7 in the gut and lung); the source of IgA was most probably the yolk, as the plasma was devoid of IgA till D7 post-hatch. The levels of maternal IgA decreased with time, but were still easily determined at the onset of endogenous IgA production following maturation of the adaptive immune system. Persistence of maternal IgA in the gut was enabled by goblet cell up-take by a yet un-described mechanism, and its consequent release in a mucin-like layer on enterocyte apical surfaces.     

实验动物弓形虫几种检测方法的应用评价研究

目的:评价检测实验动物弓形虫的2种方法。方法:应用间接免疫荧光法(IFA)、间接酶联染色法(IEA)、间接酶联免疫吸附法(ELISA)对实验动物的血清样品进行检测,以间接免疫荧光(IFA)法作为检测弓形虫的标准方法,比较后2种方法的检出率、敏感性、特异性等。结果血清经IFA、IEA、ELISA检测,分别有32.73%(18/55)、41.86(36/86)和6.34%(4/63)阳性;IFA、IEA两种方法检测血清的阳性率之间无显著性差异(P>0.05),IFA与ELISA两种方法检测血清的阳性率之间有显著性差异(P<0.05)。IEA、ELISA敏感性和特异性分别为100%与86.48%,13.33%与100%;经独立性检验,IFA与IEA两种方法存在关联性(P<0.05),IFA与ELISA两种检测方法相互独立(P>0.05)。结论:与IFA相比,IEA检测弓形虫抗体具有相似的敏感性及特异性,检出率较高,可适用于基层单位实验动物弓形虫抗体的检测;ELISA检测弓形虫抗体敏感性不高,检出率低,仍需进一步探讨和完善。