Development and characterization of SARS-CoV-2 variant-neutralizing monoclonal antibodies.

Vaccination and administration of monoclonal antibody cocktails are effective tools to control the progression of infectious diseases and to terminate pandemics such as COVID-19. However, the emergence of SARS-CoV-2 mutants with enhanced transmissibility and altered antigenicity requires broad-spectrum therapies. Here we developed a panel of SARS-CoV-2 specific mouse monoclonal antibodies (mAbs), and characterized them based on ELISA, Western immunoblot, isotyping, and virus neutralization. Six neutralizing mAbs that exhibited high-affinity binding to SARS-CoV-2 spike protein were identified, and their amino acid sequences were determined by mass spectrometry. Functional assays confirmed that three mAbs, F461G11, F461G15, and F461G16 neutralized four variants of concern (VOC): B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.2 (delta) These mAbs are promising candidates for COVID-19 therapy, and understanding their interactions with virus spike protein should support further vaccine and antibody development.

Development and characterization of monoclonal antibodies for rapid detection of Acinetobacter baumannii.

Recently Acinetobacter baumannii has emerged as a dominant form of nosocomial infections worldwide. As such, diagnostic tools are urgently needed. Reported here is the development/characterization of two mouse monoclonal antibodies (MAbs), F241G3sc2 and F241G6sc2, against A. baumannii ATCC 19606 with clinical strain cross-reactivity. Specifically, both MAbs cross-reacted with 33% of tested A. baumannii clinical strains, without cross-reactivity against other tested species of Acinetobacter. However, further testing with additional clinical strains and species is needed. Taken together, these results demonstrate both antibodies specifically target A. baumannii. With lower limits of detection at 0.32 ng/µL, both MAbs proved highly sensitive. Co-immunoprecipitation assays/LC-MS/MS, dot blots, and ELISAs eliminated the most abundant surface protein, outer membrane protein A (OmpA), as a protein target. However, since most of the proteins within the A. baumannii proteome are uncharacterized, exact protein targets could not be confirmed. Overall, these MAbs demonstrate practical applications, including ELISA, Western blot analysis, and co-IP assays, suitable to address the urgency for rapid detection tools required for A. baumannii research.

Screening hybridomas for cell surface antigens by high-throughput homogeneous assay and flow cytometry.

Described herein are methods for the successful screening of monoclonal antibodies (mAbs) of the desired specificities via high-throughput (HTP) homogeneous assay and flow cytometry. We present a combination of screening techniques that allow the scientist to efficiently eliminate nontarget-specific antibody as soon as possible. This compilation of protocols will enable researchers with basic immunology skills to make decisions regarding the design of screening algorithms for the generation of mAbs. Although we have provided an informative overview of both HTP homogeneous assay and flow cytometry, it is imperative for the beginner to acquire fundamental knowledge on how both of these technologies work so as to use these screening strategies effectively.

Development of neutralizing monoclonal antibodies against the pandemic H1N1 virus (2009) using plasmid DNA immunogen.

Reported here is the development and characterization of eighteen mouse monoclonal antibodies (MAbs) against the 2009 pandemic H1N1 influenza virus (A/Mexico/InDRE4487/2009). To our knowledge, this is the first report on pandemic (pH1) H1N1 MAbs developed using plasmid DNA encoding the viral surface glycoprotein, hemagglutinin (HA). All eighteen MAbs were specific for A/Mexico/InDRE4487/2009 HA. Ten MAbs were found to cross-react with A/Swine/Indiana/81 using a dot blot assay. However, there was no cross-reactivity detected against any other strains of influenza A viruses despite screening against all 16 hemagglutinin subtypes. Examination of these MAbs identified individual antibodies suitable for use in several practical applications including ELISA, immunoblot and immunofluorescence assays. Analysis of the kinetics of each MAb revealed significant binding affinities (K(D)<10(-8) M) confirming the antibodies are highly specific for A/Mexico/InDRE4487/2009 HA. Functional analysis demonstrated the panel of MAbs included antibodies with HA inhibition and virus neutralization activities. Not all MAbs inhibited hemagglutination or neutralized the virus. Furthermore, the panel of MAbs was not found to be cross-reactive against additional strains tested in hemagglutination inhibition assays. Finally, the MAbs were tested in competitive ELISA (cELISA) using reference serum antibodies developed against different clusters of H1 (pH1, α, ß, γ, and δ). The developed MAbs outcompeted serum antibodies of pH1 in 16/18, 15/18 (γ), 3/18 (α), 2/18 (δ1) and 1/18 (ß) samples. Overall, this panel of MAbs proved specific and highly sensitive for A/Mexico/InDRE4487/2009 HA and could potentially serve as immunodiagnostic tools for the rapid detection of this specific strain of influenza virus.

Successful treatment of ebola virus-infected cynomolgus macaques with monoclonal antibodies.

Ebola virus (EBOV) is considered one of the most aggressive infectious agents and is capable of causing death in humans and nonhuman primates (NHPs) within days of exposure. Recent strategies have succeeded in preventing acquisition of infection in NHPs after treatment; however, these strategies are only successful when administered before or minutes after infection. The present work shows that a combination of three neutralizing monoclonal antibodies (mAbs) directed against the Ebola envelope glycoprotein (GP) resulted in complete survival (four of four cynomolgus macaques) with no apparent side effects when three doses were administered 3 days apart beginning at 24 hours after a lethal challenge with EBOV. The same treatment initiated 48 hours after lethal challenge with EBOV resulted in two of four cynomolgus macaques fully recovering. The survivors demonstrated an EBOV-GP-specific humoral and cell-mediated immune response. These data highlight the important role of antibodies to control EBOV replication in vivo, and support the use of mAbs against a severe filovirus infection.

Development and characterization of neutralizing monoclonal antibodies against the pandemic H1N1 virus (2009).

The 2009 H1N1 influenza pandemic was a major international public health crisis which caused considerable morbidity and mortality worldwide. The goal of this study was to produce anti-H1 monoclonal antibodies (MAbs) for improving diagnostic immunological assays and to develop potential immunotherapeutics. Nine MAbs were produced after immunizing mice with recombinant hemagglutinin (HA) protein from A/California/06/09. Two spleenocyte myeloma fusions yielded 1588 hybridoma cultures. After screening the hybridoma culture supernatants for antibody reactivity to rHA, nine clones were selected for further characterization. Cross-reactivity studies of the anti-rHA antibodies against a panel of influenza viruses (H1-H16) revealed eight out of nine MAbs were specific to the pandemic H1 subtype, except for MAb F256G2sc1 which also cross-reacted with H5 subtype virus. All MAbs were of the IgG1κ isotype, except F256G2sc1 which was IgG2aκ. The anti-rHA MAbs had binding affinities to rHA that ranged from a K(D) (disassociation constant) of 1.34×10(-9)M (F255G7sc1) to the weakest affinity of 4.60×10(-8)M (F255G4sc1). Interestingly, in a plaque reduction neutralization assay, all MAbs except F255G3sc1 demonstrated neutralizing ability. Furthermore, all MAbs except F255G3sc1 and F255G9sc1 exhibited anti-hemagglutinin activity against pandemic H1N1 viruses, but not against classical North American swine influenza viruses of the same subtype. Immunofluorescence assay (IFA) demonstrated that all MAbs except F255G1sc1 and F255G3sc1 were able to detect 2009 pandemic H1N1 (2009) virus- infected MDCK cells. The MAbs were also evaluated for potential use in competitive ELISA (cELISA), and with the exception of F255G3sc1, all MAbs showed competitive activity with serum collected from pigs infected with pandemic H1N1 virus (2009). The developed MAbs have demonstrated utility as immunodiagnostic and research reagents, and their neutralizing capabilities also hold potential for designing antiviral drugs against pandemic influenza.

EGFR/erB-1, HER2/erB-2, CK7, LP34, Ki67 and P53 expression in preneoplastic lesions of bronchial epithelium: an immunohistochemical and genetic study.

A prognostic interpretation of preneoplastic lesions would have impact in bronchial carcinoma early diagnosis and through the study of Erb-B family receptors as they have an important role in lung carcinogenesis. The existence of drugs as tyrosine kinase inhibitors stressed the importance of studying gene alterations for selected chemoprevention schemes and characterization of carcinogenesis. Bronchial preneoplastic lesions were characterized by immunohistochemistry using the antibodies LP34 (high weigh molecular cytokeratin), CK7, chromogranin A, Ki67, p53, C-erbB-2 and EGFR. HER2 and EGFR gene copy number was also evaluated by fluorescent in situ hybridization in those lesions. The expected results defined the origin cell for basal cell hyperplasia and squamous metaplasia as adaptative lesions and dysplasia. By known experiences and published data, beyond the stem cell, the spectral evolution of bronchial preneoplastic lesions was demonstrated by characterizing basal cells (LP34) and their neoplastic potentiality. Dysplasias showed a higher expression of EGFR, Ki67 and p53 with a stepwise increase with the gravity of the respective grading. C-erbB-2 immunohistochemical overexpression was a rare event in preneoplastic lesions. Polysomy was the main mechanism for EGFR and HER2/neu higher gene copy number and together with increased proliferation index (Ki67) will account to preview bronchial carcinogenesis.

Production and characterization of a monoclonal antibody to Francisella tularensis lipopolysaccharide.

Having the capacity to detect and identify pathogens that can be employed in a bioterror attack is critical from both a public health and defence perspective. Immunodiagnostic assays are useful tools for enhancing such detection capabilities. In order to develop an immunodiagnostic assay for the detection of Francisella tularensis, a murine monoclonal antibody (MAb) was developed, using the live vaccine strain (LVS) of F. tularensis as the inoculating antigen. A single MAb, F94G2-1, which is specific for the lipopolysaccharide (LPS) of this bacterium was developed and characterized. An indirect ELISA using purified LPS was effective in determining reactivity of the MAb against its target. An immunodotblot and a manually printed antigen microarray were also tested as suitable detection methods. Both assays showed that MAb F94G2-1 has excellent specificity for F. tularensis LPS and demonstrate the utility of using the same MAb in a variety of immunodiagnostic applications.

Structure-activity studies with cytotoxic anthrapyrazoles.

Anthrapyrazoles have been investigated as cancer chemotherapeutic agents. The mechanism of action of these compounds is thought to involve inhibition of DNA topoisomerase II. A structure-activity study was carried out to determine the in vitro cytotoxic activity of nine novel anthrapyrazoles against human breast carcinoma, head and neck squamous cell carcinoma and leukemia cells, and against Chinese hamster ovary cells. The activity of these anthrapyrazole analogues was compared with that of two clinically tested anthrapyrazoles, losoxantrone and piroxantrone. Inhibition of topoisomerase II as a mechanism of action for the analogues was also investigated. The cytotoxic activity of the analogues was determined in vitro by MTT cell growth inhibition assay and inhibition of catalytic topoisomerase II activity by each compound was measured using a fluorometric DNA decatenation assay. All of the anthrapyrazole analogues inhibited the growth of the four cell lines with IC50 values that ranged from 0.1 to 45.2 microM. Losoxantrone was the most potent of the anthrapyrazole analogues studied. A tertiary amine in the basic side chain at N-2 increased the cytotoxic activity compared with a secondary amine in this side chain for many of the analogues, but not if there was a basic side chain at the C-5 position. A chlorine substituent on the basic side chain at N-2 did not have a consistent effect on activity. Moving the position of a chlorine substituent from C-5 to C-7 or introducing a basic side chain at C-5 did not have a consistent effect on cytotoxic activity. Anthrapyrazole analogues showed a broad range of activity for inhibiting topoisomerase II decatenation activity. Losoxantrone and piroxantrone were the most potent inhibitors of topoisomerase II activity. There was no significant correlation between the cytotoxic activity of the anthrapyrazole analogues and their ability to inhibit decatenation by topoisomerase II.

Role of NQO1 polymorphisms as risk factors for squamous cell carcinoma of the head and neck.

A case: control study was carried out to determine if inactivating polymorphisms of the NQO1 gene at bases 609 and 465 are associated with altered risk of developing squamous cell carcinoma of the head and neck (SCCHN). Genotyping was carried out by PCR RFLP analysis on whole blood samples. The frequency of the inactive 609T and active 609C forms, and the inactive 465T and active 465C forms, of NQO1 were compared in patient and control groups by a logistic regression analysis and odds ratios (ORs) were calculated. Participants were stratified by tobacco and alcohol use, and genotype distributions in these sub-groups were compared. There were no significant differences in genotype distribution between SCCHN patients and the control population for the base 609 or 465 polymorphisms. There were also no significant differences in genotype distributions between patient and control groups for tobacco and/or alcohol users and non-users. Genotype distributions were similar for SCCHN patients at all disease sites with the exception of the nasopharynx where there was a higher incidence of the 609C:609T and 609T:609T genotypes. These results suggest that individuals having either 609T or 465T alleles generally do not have an altered risk of developing SCCHN.

Identification of networks of sexually transmitted infection: a molecular, geographic, and social network analysis.

BACKGROUND: Despite widespread efforts to control it, Chlamydia trachomatis remains the most frequently diagnosed bacterial sexually transmitted infection (STI). Analysis of sexual networks has been proposed as a novel tool for control of and research into STI. In the present study, we combine molecular genotype data, analysis of geographic clusters, and sociodemographic descriptors to facilitate analysis of large sexual networks. METHODS: Individual chlamydia genotypes found in Manitoba, Canada, were analyzed to identify geographic clusters, and the identified clusters were further characterized by statistical analysis of sociodemographic variables. RESULTS: A total of 10 geographic clusters of chlamydia-genotype infection were identified. Clusters in Winnipeg showed no or little geographic overlap and could be further differentiated on the basis of the sociodemographic characteristics of the individuals within a cluster. Several clusters in northern Manitoba overlapped geographically but, nonetheless, could be differentiated on the basis of the sociodemographic characteristics of the infected individuals. CONCLUSIONS: On the basis of results of the combined analyses, each geographic cluster appeared to represent a relatively distinct transmission network within the larger sexual network. The geographic analysis of the molecular data provided a basis for establishment of potential epidemiological connections between small groups of unlinked individuals. Analytic approaches of the type described here would help to decipher the patterns that exist within large social network data sets and would be applicable to many types of infectious agents.

Chlamydia trachomatis omp1 genotypic diversity and concordance with sexual network data.

Sexual and social network analysis have been proposed as novel sexually transmitted disease control and research tools. Here, the concordance between chlamydia genotype data and a large sexual network constructed from routinely collected contact tracing data was examined. A sexual network was constructed for Manitoba, Canada, from province-wide contact tracing data. Positive chlamydia specimens from the same time period were collected and genotyped by omp1 DNA sequencing. A high degree of concordance was found between transmission events, on the basis of molecular data, and proposed transmission events, on the basis of sexual network data. Discordant results appeared to occur when a portion of the network contained potential core group members or in areas where contact tracing is difficult to carry out. The agreement between the molecular and epidemiologic data suggests that the use of routine contact tracing data is a valid approach for the construction of sexual networks.

Coliformes totais e fecais e caracterizaçäo dos coliformes fecais em queijo tipo coalho comercializado em Joäo Pessoa - Pb / Total and faecal coliforms and characterization of faecal coliforms in cheese type clot commercialized in Joao Pessoa - Pb

Em 50 amostras de queijo tipo "coalho" coletadas em supermercados, mercearias, feiras-livres e padarias foram efetuadas as determinaçöes do Número Mais Povável (NMP) de bactérias coliformes totais e fecais com a finalidade de verificar as suas condiçöes microbiológicas e compará-las com os padröes recomendados. Culturas de coliformes em número de 200, isoladas do produto foram testadas quanto à capacidade de produzir gases quando incubadas em Caldo lactose e submetidas ao IMViC. Todas as amostras mostraram-se positivas para bactérias coliformes totais com os NMPs variando de 1,7 x 10(*3) a igual e/ou maior que 1,6 x 10(*5)/g. Com relaçäo ao NMP de coliformes fecais os resultados obtidos mostraram-se compreendidos entre zero e igual e/ou maior que 1,6 x 10(*5)/g sendo que 46 (92%) revelaram-se com número igual ou superior a 10(*3)/g. Das cepas isoladas foram detectadas em 61% das amostras coliformes fecais, sendo 24% de E.coli (21,5% do tipo I), 20,5% de Intermédios (11% do tipo II); 9,5% de Enterobacteraerogenes (5% do tipo II) e 7% de coliformes irregulares (4% do tipo II). Tais constataçöes evidenciaram a ocorrência de contaminaçäo inclusive por microrganismos de origem fecal