RESUMEN

Background: Pharyngeal infection is more difficult to diagnose and treat than genital or rectal infection and can act as a reservoir for gonococcal infection. We determined the prevalence of pharyngeal gonorrhea in Korean men with urethritis and analyzed the molecular characteristics and antimicrobial susceptibility of the isolates. Methods: Seventy-two male patients with symptoms of urethritis who visited a urology clinic in Wonju, Korea, between September 2016 and March 2018 were included. Urethral and pharyngeal gonococcal cultures, antimicrobial susceptibility testing, Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST), and multiplex real-time PCR (mRT-PCR) were performed. Results: Among the 72 patients, 59 tested positive for gonococcus by mRT-PCR. Of these 59 patients, 18 (30.5%) tested positive in both the pharynx and urethra, whereas 41 tested positive only in the urethra. NG-MAST was feasible in 16 out of 18 patients and revealed that 14 patients had the same sequence types in both urethral and pharyngeal specimens, whereas two patients exhibited different sequence types between the urethra and pharynx. Of the 72 patients, 33 tested culture-positive. All patients tested positive only in urethral specimens, except for one patient who tested positive in both. All culture-positive specimens also tested positive by mRT-PCR. All isolates were susceptible to azithromycin and spectinomycin, but resistance rates to ceftriaxone and cefixime were 2.9% and 14.7%, respectively. Conclusions: The prevalence of pharyngeal gonorrhea in Korean men with gonococcal urethritis is as high as 30.5%, highlighting the need for pharyngeal screening in high-risk groups. Ceftriaxone is the recommended treatment for pharyngeal gonorrhea.

Asunto(s)

Antibacterianos , Gonorrea , Pruebas de Sensibilidad Microbiana , Neisseria gonorrhoeae , Faringe , Uretra , Uretritis , Humanos , Masculino , Gonorrea/microbiología , Gonorrea/diagnóstico , Gonorrea/epidemiología , Gonorrea/tratamiento farmacológico , Uretritis/microbiología , Uretritis/diagnóstico , Uretritis/epidemiología , República de Corea/epidemiología , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/aislamiento & purificación , Neisseria gonorrhoeae/efectos de los fármacos , Adulto , Prevalencia , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Uretra/microbiología , Persona de Mediana Edad , Faringe/microbiología , Adulto Joven , Reacción en Cadena en Tiempo Real de la Polimerasa , Farmacorresistencia Bacteriana , Enfermedades Faríngeas/microbiología , Enfermedades Faríngeas/epidemiología , Enfermedades Faríngeas/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex

RESUMEN

This "Magnum Opus" accentuates my lifelong belief that the future of science is in the interdisciplinary approach to hypotheses formulation and problem solving. Inspired by the invention of hydrogels and soft contact lenses by my mentors, my six decades of research have continuously proceeded from the synthesis of biocompatible hydrogels to the development of polymer-drug conjugates, then generation of drug-free macromolecular therapeutics (DFMT) and finally to multi-antigen T cell hybridizers (MATCH). This interdisciplinary journey was inspiring; the lifetime feeling that one is a beginner in some aspects of the research is a driving force that keeps the enthusiasm high. Also, I wanted to illustrate that systematic research in one wide area can be a life-time effort without the need to jump to areas that are temporarily en-vogue. In addition to generating general scientific knowledge, hydrogels from my laboratory have been transferred to the clinic, polymer-drug conjugates to clinical trials, and drug-free macromolecular systems have an excellent potential for personalizing patient therapies. There is a limit to life but no limit to imagination. I anticipate that systematic basic research will contribute to the expansion of our knowledge and create a foundation for the design of new paradigms based on the comprehension of mechanisms of physiological processes. The emerging novel platform technologies in biomaterial-based devices and implants as well as in personalized nanomedicines will ultimately impact clinical practice.

Asunto(s)

Materiales Biocompatibles , Hidrogeles , Polímeros , Hidrogeles/química , Humanos , Materiales Biocompatibles/química , Polímeros/química , Animales , Interacciones Hidrofóbicas e Hidrofílicas , Sustancias Macromoleculares/química

RESUMEN

African swine fever is an acute and highly contagious infectious disease with a mortality rate of up to 100%. The lack of commercial vaccines and drugs is a serious economic threat to the global pig industry. Cell-mediated immunity plays an essential role in protection against viral infection. We previously reported the rational design of a T-cell-activating thermostable scaffold (RPT) for antigen delivery and improved cellular immunity. We conjugated antigens P30, P54, P72, CD2 V, and CP312R to RPT, using a SpyCatcher/SpyTag covalent attachment strategy to construct nanovaccines (multiantigens-RPT). Multiantigens-RPT exhibited significantly higher thermal, storage, and freeze-thaw stability. The specific antibodies IgG and IgG2a of the multiantigen-RPT-immunized were higher than the antigens cocktail-immunized by approximately 10-100 times. ELISpot demonstrated that more IFN-γ-secreting cells were produced by the multiantigen-RPT-immunized than by the antigens cocktail-immunized. Delivery of the multiantigen nanovaccine by a T-cell-activating scaffold induced strong humoral and cellular immune responses in mice and pigs and is a potentially useful candidate vaccine for the African swine fever virus.

Asunto(s)

Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Porcinos , Animales , Ratones , Fiebre Porcina Africana/prevención & control , Linfocitos T , Nanovacunas , Adyuvantes Inmunológicos

RESUMEN

Baylisascaris schroederi is among the most severe intestinal nematodes affecting giant pandas. Developing effective and secure vaccines can be used as a novel strategy for controlling repeated roundworm infection and addressing drug resistance. In our previous study, three recombinant antigens (rBsHP2, rBsGAL, and rBsUP) exhibited promising effects against B. schroederi infection in the mice model. This study extends the findings by formulating four-form cocktail vaccines (GAL+UP, HP2+UP, GAL+HP2, and GAL+HP2+UP) using three B. schroederi recombinant antigens to improve protection in mice further. Additionally, the protective differences after immunizing mice with different doses of cocktail antigens (150 µg, 100 µg, and 50 µg) were analyzed. Administration of rBs(GAL+UP), rBs(HP2+UP), rBs(GAL+HP2), and rBs(GAL+HP2+UP) significantly reduced liver and lung lesions, along with a decrease in L3 larvae by 83.7%, 82.1%, 76.4%, and 75.1%, respectively. These vaccines induced a Th1/Th2 mixed immunity, evidenced by elevated serum antibody levels (IgG, IgG1, IgG2a, IgE, and IgA) and splenocyte cytokines [interferon gamma (IFN-γ), interleukin (IL)-5, and IL-10]. Furthermore, varying cocktail vaccine dosages did not significantly affect protection. The results confirm that a 50 µg rBs(GAL+UP) dosage holds promise as a better candidate vaccine combination against B. schroederi infection, providing a basis for developing the B. schroederi vaccine.

Asunto(s)

Ascaridoidea , Vacunas , Animales , Ratones , Proteínas Recombinantes , Antígenos Helmínticos/genética , Ascaridoidea/genética , Ratones Endogámicos BALB C

RESUMEN

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (EP), leading to a mild and chronic pneumonia in swine. Relative control has been attained through active vaccination programs, but porcine enzootic pneumonia remains a significant economic challenge in the swine industry. Cellular immunity plays a key role in the prevention and control of porcine enzootic pneumonia. Therefore, the development of a more efficient vaccine that confers a strong immunity against M. hyopneumoniae is necessary. In this study, a multi-antigen chimera (L9m6) was constructed by combining the heat-labile enterotoxin B subunit (LTB) with three antigens of M. hyopneumoniae (P97R1, mhp390, and P46), and its immunogenic and antigenic properties were assessed in a murine model. In addition, we compared the effect of individual administration and multiple-fusion of these antigens. The chimeric multi-fusion vaccine induced significant cellular immune responses and high production of IgG and IgM antibodies against M. hyopneumoniae. Collectively, our data suggested that rL9m6 chimera exhibits potential as a viable vaccine candidate for the prevention and control of porcine enzootic pneumonia.

RESUMEN

Current unprecedented mpox outbreaks in non-endemic regions represent a global public health concern. Although two live-attenuated vaccinia virus (VACV)-based vaccines have been urgently approved for people at high risk for mpox, a safer and more effective vaccine that can be available for the general public is desperately needed. By utilizing a simplified manufacturing strategy of mixing DNA plasmids before transcription, we developed two multi-antigen mRNA vaccine candidates, which encode four (M1, A29, B6, A35, termed as Rmix4) or six (M1, H3, A29, E8, B6, A35, termed as Rmix6) mpox virus antigens. We demonstrated that those mpox multi-antigen mRNA vaccine candidates elicited similar potent cross-neutralizing immune responses against VACV, and compared to Rmix4, Rmix6 elicited significantly stronger cellular immune responses. Moreover, immunization with both vaccine candidates protected mice from the lethal VACV challenge. Investigation of B-cell receptor (BCR) repertoire elicited by mpox individual antigen demonstrated that the M1 antigen efficiently induced neutralizing antibody responses, and all neutralizing antibodies among the top 20 frequent antibodies appeared to target the same conformational epitope as 7D11, revealing potential vulnerability to viral immune evasion. Our findings suggest that Rmix4 and Rmix6 from a simplified manufacturing process are promising candidates to combat mpox.

Asunto(s)

Mpox , Orthopoxvirus , Animales , Ratones , Anticuerpos Antivirales , Orthopoxvirus/genética , Proteínas del Envoltorio Viral , Anticuerpos Neutralizantes , Virus Vaccinia/genética

RESUMEN

The emergence of Neisseria gonorrhoeae isolates displaying resistance to antimicrobials, in particular to ceftriaxone monotherapy or ceftriaxone plus azithromycin, represents a global public health concern. This study aimed to analyze the trend of antimicrobial resistance in a 7-year isolate collection retrospective analysis in Italy. Molecular typing on a subsample of gonococci was also included. A total of 1,810 culture-positive gonorrhea cases, collected from 2013 to 2019, were investigated by antimicrobial susceptibility, using gradient diffusion method, and by the N. gonorrhoeae multiantigen sequence typing (NG-MAST). The majority of infections occurred among men with urogenital infections and 57.9% of male patients were men who have sex with men. Overall, the cefixime resistance remained stable during the time. An increase of azithromycin resistance was observed until 2018 (26.5%) with a slight decrease in the last year. In 2019, gonococci showing azithromycin minimum inhibitory concentration above the EUCAST epidemiological cutoff value (ECOFF) accounted for 9.9%. Ciprofloxacin resistance and penicillinase-producing N. gonorrhoeae (PPNG) percentages increased reaching 79.1% and 18.7% in 2019, respectively. The most common sequence types identified were 5,441, 1,407, 6,360, and 5,624. The predominant genogroup (G) was the 1,407; moreover, a new genogroup G13070 was also detected. A variation in the antimicrobial resistance rates and high genetic variability were observed in this study. The main phenotypic and genotypic characteristics of N. gonorrhoeae isolates were described to monitor the spread of drug-resistant gonorrhea.

Asunto(s)

Gonorrea , Minorías Sexuales y de Género , Humanos , Masculino , Femenino , Antibacterianos/farmacología , Neisseria gonorrhoeae , Gonorrea/tratamiento farmacológico , Gonorrea/epidemiología , Ceftriaxona/farmacología , Ceftriaxona/uso terapéutico , Azitromicina/farmacología , Epidemiología Molecular , Estudios Retrospectivos , Homosexualidad Masculina , Farmacorresistencia Bacteriana/genética , Pruebas de Sensibilidad Microbiana

RESUMEN

To gain world-wide control over COVID-19 pandemic, it is necessary to have affordable and accessible vaccine and monoclonal antibody technologies across the globe. In comparison to the western countries, Asian and African countries have less percentage of vaccination done which warrants urgent attention. Global manufacturer production capacities, dependency on advanced nations for the supply of vaccines or the raw material, national economy, limited research facilities, and logistics could be the factors. This review article elaborates the existing therapeutic and prophylactic strategies available for COVID-19, currently adopted vaccine and monoclonal antibody platforms for SARS-CoV-2 along with the approaches to bridge the gap prevailing in the challenges faced by low- and middle-income countries. We believe adoption of yeast-derived P. pastoris technology can help in developing safe, proven, easy to scale-up, and affordable recombinant vaccine or monoclonal antibodies against SARS-CoV-2. This platform has the advantage of not requiring a dedicated or specialized facility making it an affordable option using existing manufacturing facilities, without significant additional capital investments. Besides, the technology platform of multiantigen vaccine approach and monoclonal antibody cocktail will serve as effective weapons to combat the threat posed by the SARS-CoV-2 variants. Successful development of vaccines and monoclonal antibodies using such a technology will lead to self-sufficiency of these nations in terms of availability of vaccines and monoclonal antibodies.

Asunto(s)

COVID-19 , Vacunas , Anticuerpos Monoclonales/uso terapéutico , COVID-19/prevención & control , Países en Desarrollo , Humanos , Pandemias/prevención & control , SARS-CoV-2

RESUMEN

Gonorrhea is a sexually transmitted infection occurring worldwide. Antimicrobial resistance (AMR) surveillance in Neisseria gonorrhoeae and associated molecular epidemiological studies are crucial to ascertain the spread of antibiotic-resistant and developing the local treatment guidelines. This study was performed to determine the antimicrobial susceptibility testing (AST) and molecular epidemiology of N. gonorrhoeae isolates in Tehran, Iran. During 1 July 2018-30 July 2020, a total of 500 urogenital (468 endocervical, 32 urethral) swabs were collected from patients with signs and symptoms of genitourinary infections presenting to two women's hospitals and one health center located center and south of Tehran. Specimens were cultured and examined for the presence of N. gonorrhoeae isolates by biochemical tests. MIC Test Strip determined the MICs of ceftriaxone, azithromycin, and ciprofloxacin. Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) was also performed. A total of 38 N. gonorrhoeae isolates were identified. The proportions of resistant N. gonorrhoeae isolates were as follows: ceftriaxone (MIC ≥0.125 µg/mL) 10.5% (4/38), azithromycin (MIC >1 µg/mL) 34% (13/38), and ciprofloxacin (MIC ≥1 µg/mL) 31.5% (12/38). In total, 25 different NG-MAST STs were identified. The STs comprised 1-4 isolates each, and the predominant ST was ST266 (n = 4). Our study demonstrates a diverse gonococcal population with high rates of resistance to azithromycin and evidence of resistance to ceftriaxone. The results have potential implications for antibiotic choice for the gonococcal treatment and highlight the need to broaden gonococcal AMR monitoring in Iran.

Asunto(s)

Gonorrea , Neisseria gonorrhoeae , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Azitromicina/farmacología , Azitromicina/uso terapéutico , Ceftriaxona/farmacología , Ceftriaxona/uso terapéutico , Ciprofloxacina/farmacología , Ciprofloxacina/uso terapéutico , Farmacorresistencia Bacteriana , Femenino , Gonorrea/diagnóstico , Gonorrea/tratamiento farmacológico , Gonorrea/epidemiología , Humanos , Irán/epidemiología , Pruebas de Sensibilidad Microbiana

RESUMEN

Bovine Tuberculosis (bTB) is a chronic disease caused by Mycobacterium bovis, affecting cattle and other mammalian species, such as pigs. In the present work, we developed a novel multi-antigen assay (The TB-Luminex multiplex test) to diagnose bTB in pig sera. Moreover, we investigated the seroreactivity to the different antigens employed (MPB83, MPB70, CFP10 and ESAT6) and the possible correlation with bTB lesions distribution in the positive pigs. The serum samples were collected from 59 bTB positive pigs and 186 pigs reared in an officially Tuberculosis free area. Sera were processed according to an optimized protocol for the detection of antibodies by a multiantigen assay using Luminex technology. The positive group showed visible lesions with localized (54.2%) or generalized (45.8%) distribution. Culture confirmed the infection in 62.7% of the cases, and histopathology and intra-vitam assays were used as additional confirmatory tests. Within the set of antigens tested, the immunodominant was MPB83 (positive in 94.9% of the affected pigs), followed by CFP10, MPB70 and ESAT6 (positivity shown in 81.3%, 67.8% and 25.4% of the positive pigs tested, respectively). The best antigens combination was MPB83/CFP10, with a 96.6% sensitivity and 96.8% specificity. Overall, the test showed high sensitivity (98.3% and 86.4%) and specificity (96.2% and 97.8%), if sera were considered positive according to the positivity to a single antigen or at least two antigens, respectively. The TB-Luminex multiplex test results did not give significantly different outcomes according to lesions distribution. Given the present study results, the TB-Luminex multiplex test is a reliable test capable of detecting bTB in most infected pigs with good Se and Sp, regardless of the stage of the disease. In conclusion, multi-antigen tests can be used as individual tests and screening tools for domestic and wild suids within bTB eradication programs.

Asunto(s)

Mycobacterium bovis , Tuberculosis Bovina , Tuberculosis , Animales , Anticuerpos Antibacterianos , Bovinos , Mamíferos , Pruebas Serológicas/métodos , Pruebas Serológicas/veterinaria , Porcinos , Tuberculosis/diagnóstico , Tuberculosis/veterinaria , Tuberculosis Bovina/diagnóstico

RESUMEN

Anti­CD19 chimeric antigen receptor (CAR)­T cell therapy against refractory B­cell malignancies shows excellent therapeutic effects. However, there are some obstacles to be overcome in this treatment. Since current CAR­T cells target a single cell­surface protein on tumor cells, the CAR­T cells also attack normal cells expressing the protein. This is one of the major adverse effects of this therapy. To improve target­cell­specificity of this therapy, we established a novel CAR system, in which T­cell activation was controlled by expression patterns of proteins on target cells. Our novel CAR­T cells had two distinct CARs consisting of a 'Signal­CAR', recognizing a protein on tumor cells, and a 'Scissors­CAR', recognizing another protein on normal cells. The signal­CAR had a peptide sequence which was cleaved by the Scissors­CAR, and functional domains for cellular activation. The Scissors­CAR had a protease domain that cleaved its recognition peptide sequence in the Signal­CAR. When tumor cells expressed only the protein recognized by the Signal­CAR, the tumor cells were attacked. By contrast, normal cells expressing both the proteins induced inactivation of the Signal­CAR through cleavage of the recognition site when getting in contact with the CAR­T cells. To establish this system, we invented a Scissors­CAR that was dominantly localized on cell membranes and was activated only when the CAR­T cells were in contact with the normal cells. Using a T­cell line, Jurkat, and two proteins, CD19 and HER2, as target proteins, we showed that the anti­CD19­Signal­CAR was cleaved by the anti­HER2­Scissors­CAR when the CAR­T cells were co­cultivated with cells expressing both the proteins, CD19 and HER2. Furthermore, we demonstrated that primary CAR­T cells expressing both the CARs showed attenuated cytotoxicity againsT cells with both the target proteins. Our novel system would improve safety of the CAR­T cell therapy, leading to expansion of treatable diseases by this immunotherapy.

Asunto(s)

Receptores Quiméricos de Antígenos , Antígenos CD19/genética , Antígenos CD19/metabolismo , Proteínas de la Membrana/metabolismo , Péptido Hidrolasas/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/metabolismo

RESUMEN

Ticks are blood sucking ectoparasite that transmit several pathogens to humans and animals. Tick management focusing on use of chemicals has several drawbacks including development of multi-acaricide resistant tick populations. To minimize the use of chemicals on animals and on the environment, immunization of natural hosts is considered a viable component of Integrated Tick Management System. Most of the tick vaccine trials are focused on single antigen immunization directed against homologous challenge. From commercial point of view, vaccination against one given tick species is not a feasible option. In this context, multi-antigen vaccines comprising of candidate antigens of multiple tick species or both ticks and tick-borne pathogens have commercial potential. Different strategies are considered for the development of multi-antigen tick and/or tick-borne pathogen vaccines. Further, the efficacy of vaccine can be improved by adopting the 'omics' tools and techniques in selection of novel antigens and efficient delivery like Lipid Nano Particle (LNP)-mRNA vaccines, viral vector vaccine, live vector vaccine etc. into the host. The subject has been reviewed to address the current status of multi antigen tick vaccines and formulations of the future strategies for the control of TTBDs of human and animals.

Asunto(s)

Acaricidas , Infestaciones por Garrapatas , Enfermedades por Picaduras de Garrapatas , Garrapatas , Vacunas , Animales , Antígenos , Humanos , Infestaciones por Garrapatas/prevención & control , Enfermedades por Picaduras de Garrapatas/prevención & control

RESUMEN

This work aimed to study penA gene polymorphisms in clinical isolates of Neisseria gonorrhoeae collected in Russia in 2018-2019 and the contribution of the penA allele type to susceptibility to ß-lactam antibiotics. A total of 182 isolates were analyzed. penA allele types were determined by sequencing, and the minimum inhibitory concentrations (MICs) of benzylpenicillin and ceftriaxone were measured. The influence of genetic factors on MICs was evaluated by regression analysis. All isolates were susceptible to ceftriaxone, and 40.1% of isolates were susceptible to penicillin. Eleven penA allele types were identified. The mosaic type XXXIV penA allele and the Gly120Lys substitution in PorB made the greatest contributions to increasing the ceftriaxone MIC; the presence of the blaTEM plasmid, Gly120Asp, Ala121Gly/Asn substitutions in PorB, and the adenine deletion in the promoter region of the mtrR gene caused an increase in the penicillin MIC. Among 61 NG-MAST types identified, the most frequent were types 228, 807, 9486, 1993, and 6226. A link between penA alleles and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) types was established. Resistance to two groups of ß-lactam antibiotics was associated with non-identical changes in penA alleles. To prevent the emergence of ceftriaxone resistance in Russia, NG-MAST genotyping must be supplemented with penA allele analysis.

RESUMEN

OBJECTIVES: The goal of this work was to assess the genetic diversity of Neisseria gonorrhoeae isolates in Russia and Europe and to compare the distribution of the N. gonorrhoeae multi-antigen sequencing types (NG-MAST) of Russian isolates with that of isolates from European countries. METHODS: NG-MAST typing was performed for 804 N. gonorrhoeae isolates collected in Russia in 2013-2018. For isolates from European countries, data from the https://pathogen.watch/collection/eurogasp2013 database were used. RESULTS: Among the isolates from Russia, 296 NG-MAST types were found. A maximum likelihood phylogenetic tree was constructed. Phylogenetic analysis revealed seven major genogroups uniting the most frequent Russian sequence types: G807, G1993, G9476, G14942, G1152, G9486, and G12531. CONCLUSIONS: The NG-MAST type distribution in Russia differed from that in European countries. Most of the Russian isolates had sequence types that were not found in Europe. Only 33% of the Russian isolates belonged to genogroups established for European countries, and the widespread European genogroup G1407 was represented by only nine isolates. Analysis of the Russian isolates belonging to phylogenetically close European genogroups indicated similarities in drug resistance, although no epidemically dangerous drug-resistant clones were found among the Russian isolates.

Asunto(s)

Variación Genética , Neisseria gonorrhoeae/genética , Antígenos Bacterianos/genética , Técnicas de Tipificación Bacteriana , Europa (Continente) , Genotipo , Humanos , Neisseria gonorrhoeae/clasificación , Neisseria gonorrhoeae/aislamiento & purificación , Filogenia , Federación de Rusia

RESUMEN

Serologic testing is the standard for laboratory diagnosis and confirmation of Lyme disease. Serodiagnostic assays to detect antibodies against Borrelia burgdorferi, the agent of Lyme borreliosis, are used for detection of infection. However, serologic testing within the first month of infection is less sensitive as patients' antibody responses continue to develop. Previously, we screened several B. burgdorferi in vivo expressed antigens for candidates that elicit early antibody responses in patients with Stage 1 and 2 Lyme disease. We evaluated patient IgM seroreactivity against 6 antigens and found an increase in sensitivity without compromising specificity when compared to current IgM second-tier immunoblot scoring. In this study, we continued the evaluation using a multi-antigen panel to measure IgM plus IgG seroreactivity in these early Lyme disease patients' serum samples. Using two statistical methods for calculating positivity cutoff values, sensitivity was 70 and 84-87%, for early acute and early convalescent Lyme disease patients, respectively. Specificity was 98-100% for healthy non-endemic control patients, and 96-100% for healthy endemic controls depending on the statistical analysis. We conclude that improved serologic testing for early Lyme disease may be achieved by the addition of multiple borrelial antigens that elicit IgM and IgG antibodies early in infection.

RESUMEN

Protection against the intraerythrocytic protozoan parasite Babesia bovis depends on both strong innate and adaptive immune response, this latter involving the presentation of parasite antigens to CD4+ T-lymphocytes by professional antigen-presenting cells. Secretion of Th1 cytokines by CD4+ T cell is also very important for isotype switching to IgG2, the best opsonising antibody isotype in cattle, to target extracellular parasites and parasite antigens displayed at the erythrocyte surface. In the field of vaccinology, heterologous prime-boost schemes combining protein-adjuvant formulations with a modified vaccinia Ankara vector expressing the same antigen have demonstrated the induction of both humoral and cellular immune responses. It has been previously demonstrated that MVA-infected dendritic cells can present antigens in the context of MHC II and activate CD4+ T cell. These results support the use of the MVA viral vector for a pathogen like Babesia bovis, which only resides within erythrocytes. In this study, 13-15-months-old Holstein-Friesian steers were immunised with a subunit vaccine as a prime and a modified vaccinia Ankara vector as a boost, both expressing a chimeric multi-antigen (rMABbo - rMVA). This antigen includes the immunodominant B and T cell epitopes of three B. bovis proteins: merozoite surface antigen - 2c (MSA - 2c), rhoptry associated protein 1 (RAP - 1) and heat shock protein 20 (HSP20). Responses were compared with the Babesia bovis live attenuated vaccine used in Argentina (R1A). Eleven weeks after the first immunisation, all bovines were challenged by the inoculation of a virulent B. bovis strain. All groups were monitored daily for hyperthermia and reduction of packed cell volume. Both the rMABbo - rMVA and R1A vaccinated animals developed high titters of total IgG antibodies and an antigen-specific Th1 cellular response before and after challenge. However, all rMABbo - rMVA steers showed clinical signs of disease upon challenge. Only the R1A live vaccine group developed an immune response associated with in vitro neutralising antibodies at a level that significantly inhibited the parasite invasion. The lack of protection observed with this recombinant formulation indicates the need to perform further basic and clinical studies in the bovine model in order to achieve the desired effectiveness. This is the first report in which a novel vaccine candidate against Babesia bovis was constructed based on a recombinant and rationally designed viral vector and evaluated in the biological model of the disease.

Asunto(s)

Babesia bovis/inmunología , Babesiosis/prevención & control , Enfermedades de los Bovinos/prevención & control , Vacunas Antiprotozoos/inmunología , Vacunación/veterinaria , Animales , Anticuerpos Neutralizantes/inmunología , Babesiosis/inmunología , Bovinos , Enfermedades de los Bovinos/inmunología , Epítopos/inmunología , Inmunidad Celular , Inmunidad Humoral , Masculino , Proteínas Recombinantes/inmunología , Células TH1/inmunología , Vacunas Atenuadas/inmunología , Virus Vaccinia/inmunología

RESUMEN

BACKGROUND: The objective of this study is to investigate gonococcal isolates using phenotypic and genotypic methods. METHODOLOGY: Sixty gonococcal isolates obtained were examined. Strains were divided into 9 resistant phenotypes: Chromosomally mediated penicillin-resistant Neisseria gonorrhoeae (CMRNGP), penicillinase-producing NG (PPNG), chromosomally mediated tetracycline-resistant NG (CMRNGT), TRNG, PPNG and TRNG, CMRNGPT, quinolone resistant NG (QRNG), Azithro R, and decreased susceptibility (DS) to ceftriaxone. These isolates were also subjected to auxotyping and NG-multi-antigen sequence typing (MAST). RESULTS: Of 60 isolates, 32 (53.33%) PPNG and only one was CMRNGP; 16 (26.66%) were CMRNGT, while 18 (30%) were TRNG. Both PPNG and TRNG found in 13 (21.66%) and none were CMRNGPT. QRNG was seen in 93.33%, 5% Azithromycin R, and 6.66% were DS to ceftriaxone. Based on auxotyping, 24 (40%) nonrequiring, 16 (26.66%) were proline requiring, 13 (21.66%) arginine requiring while 7 (11.66%) belonged to others. The most common ST was 6058 (32.5%). The discriminatory indices of antibiogram, auxotyping and NG-MAST were 0.77, 0.72, and 0.95, respectively. CONCLUSIONS: NG-MAST is the method of choice for epidemiological studies.

RESUMEN

BACKGROUND: Molecular epidemiological typing of Neisseria gonorrhoeae is crucial for monitoring the spread of resistant strains. As reference strains can be used for laboratory internal quality control, we genetically characterised the American Type Culture Collection (ATCC) gonococcal strains by Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) and porB sequence typing using public multilocus sequence typing (PubMLST). METHODS: Eight ATCC gonococcal reference strains (ATCC 19424, ATCC 31426, ATCC 35541, ATCC 43069, ATCC 43070, ATCC 49226, ATCC 49926, and ATCC 49981) from Culti-Loops (Thermo Fisher Scientific, USA) were cultured. After DNA extraction, porB and tbpB were amplified and sequenced. Sequence types (STs) and allele numbers were each determined by NG-MAST (http://www.ng-mast.net) and porB sequence typing using PubMLST (http://pubmlst.org/neisseria/porB/). RESULTS: ATCC 19424 was identified as ST 266 by NG-MAST, and as Allele 946 by PubMLST. ATCC31426 was assigned a novel ST by NG-MAST, and was assigned Allele 958 with 1.2% mismatch by PubMLST. ATCC 35541 was identified as ST 12 by NG-MAST, and as Allele 624 by PubMLST. ATCC 43069 and ATCC 43070 were both identified as ST 681 by NG-MAST, and as Allele 984 by PubMLST. ATCC 49226 was identified as ST 1572 by NG-MAST, and as Allele 2110 by PubMLST. ATCC 49926 and ATCC 49981 were both identified as ST 16496 by NG-MAST, and as Allele 928 by PubMLST. CONCLUSIONS: The ST data obtained for ATCC gonococcal reference strains by NG-MAST and porB sequence typing using PubMLST can be used for quality assurance of molecular epidemiological typing in clinical microbiological laboratories.

Asunto(s)

Alelos , ADN , Tipificación de Secuencias Multilocus , Neisseria gonorrhoeae , Neisseria , Control de Calidad

RESUMEN

The benefits of polyclonal antibodies as tools for assay-specific target discovery and detection are numerous. As the future of basic research, diagnostics and biomarker discovery is dependent on high-quality reproducible data, there is a need to understand the importance and benefits of these valuable tools. All antibody forms - polyclonal, hybridoma-based monoclonal and recombinant monoclonal - have pros and cons for development, validation and use. Yet, polyclonal antibodies are embroiled in a firestorm of controversy concerning data reproducibility. We address best practices for developing and using polyclonal antibodies, pitfalls to their use and how to avoid them, and benefits to the life science community. Eliminating their use risks overlooking the unique benefits of polyclonal antibodies as 'fit-for-purpose' life science tools.

Asunto(s)

Anticuerpos/química , Proyectos de Investigación , Afinidad de Anticuerpos , Sitios de Unión de Anticuerpos , Reproducibilidad de los Resultados

RESUMEN

Streptococcus pneumoniae has multiple protein antigens on the surface in addition to the serotype specific polysaccharide capsule antigen. Whilst the capsule antigen is the target of the polysaccharide vaccines, bacterial proteins can also act as targets for the immune system. PnuBioVax (PBV) is being developed as a multi-antigen, serotype-independent prophylactic vaccine against S. pneumoniae disease. In this study we have sought to elucidate the immune response to PBV in immunised rabbits. Sera from PBV immunised rabbits contained high levels of IgG antibodies to the PBV vaccine, and pneumococcal antigens PspA, Ply, PsaA and PiuA which are components of PBV, when compared with control sera. The PBV sera supported killing of the vaccine strain TIGR4 in an opsonophagocytic killing assay and heterologous strains 6B, 19F and 15B. In addition, incubation in PBV sera led to agglutination of several strains of pneumococci, inhibition of Ply-mediated lysis of erythrocytes and reduced bacterial invasion of lung epithelial cells in vitro. These data suggest that PBV vaccination generates sera that has multiple mechanisms of action that may provide effective protection against pneumococcal infection and give broader strain coverage than the current polysaccharide based vaccines.

Asunto(s)

Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/administración & dosificación , Vacunas Neumococicas/inmunología , Streptococcus pneumoniae/inmunología , Aglutinación , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/inmunología , Reacciones Cruzadas , Endocitosis , Femenino , Hemólisis , Inmunoglobulina G/sangre , Masculino , Viabilidad Microbiana , Proteínas Opsoninas/sangre , Fagocitosis , Conejos