RESUMEN
The COVID-19 pandemic highlighted testing inequities in developing countries. Lack of lateral flow test (LFT) manufacturing capacity was a major COVID-19 response bottleneck in low- and middle-income regions. Here we report the development of an open-access LFT for SARS-CoV-2 detection comparable to commercial tests that requires only locally available supplies. The main critical resource is a locally developed horse polyclonal antibody (pAb) whose sensitivity and selectivity are greatly enhanced by affinity purification. We demonstrate that these Abs can perform similarly to commercial monoclonal antibodies (mAbs), as well as mAbs and other pAbs developed against the same antigen. We report a workflow for test optimization using nasopharyngeal swabs collected for RT-qPCR, spiked with the inactivated virus to determine analytical performance characteristics as the limit of detection, among others. Our final prototype showed a performance similar to available tests (sensitivity of 83.3% compared to RT-qPCR, and 90.9% compared to commercial antigen tests). Finally, we discuss the possibility and the challenges of utilizing affinity-purified pAbs as an alternative for the local development of antigen tests in an outbreak context and as a tool to address inequalities in access to rapid tests.
Asunto(s)
COVID-19 , SARS-CoV-2 , SARS-CoV-2/aislamiento & purificación , Humanos , COVID-19/diagnóstico , COVID-19/virología , Anticuerpos Monoclonales , Anticuerpos Antivirales , Sensibilidad y Especificidad , AnimalesRESUMEN
Research on innovative mucosal adjuvants is essential to develop new vaccines for safe mucosal application. In this work, we propose the development of a Lactococcus lactis that expresses a variant of flagellin on its surface (FliC131*), to increase the adjuvanticity of the living cell and cell wall-derived particles (CWDP). We optimized the expression of FliC131*, and confirmed its identity and localization by Western blot and flow cytometry. We also generated CWDP containing FliC131* (CDWP-FliC131*) and evaluated their storage stability. Lastly, we measured the human TLR5 stimulating activity in vitro and assessed the adjuvanticity in vivo using ovalbumin (OVA) as a model antigen. As a result, we generated L. lactis/pCWA-FliC131*, that expresses and displays FliC131* on its surface, obtained the corresponding CWDP-FliC131*, and showed that both activated hTLR5 in vitro in a dose-dependent manner. Furthermore, CWDP-FliC131* retained this biological activity after being lyophilized and stored for a year. Finally, intranasal immunization of mice with OVA plus live L. lactis/pCWA-FliC131* or CWDP-FliC131* induced OVA-specific IgG and IgA in serum, intestinal lavages, and bronchoalveolar lavages. Our work demonstrates the potential of this recombinant L. lactis with an enhanced adjuvant effect, prompting its further evaluation for the design of novel mucosal vaccines.
Asunto(s)
Adyuvantes Inmunológicos , Flagelina , Lactococcus lactis , Ratones Endogámicos BALB C , Ovalbúmina , Receptor Toll-Like 5 , Lactococcus lactis/inmunología , Animales , Flagelina/inmunología , Flagelina/administración & dosificación , Ratones , Humanos , Ovalbúmina/inmunología , Ovalbúmina/administración & dosificación , Receptor Toll-Like 5/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Femenino , Inmunidad Mucosa/efectos de los fármacos , Inmunidad Mucosa/inmunología , Inmunización/métodos , Administración IntranasalRESUMEN
[RESUMEN]. Hacia 2022, más de 100 países habían introducido una de las cuatro vacunas autorizadas para uso mundial contra rotavirus, sin embargo, según modelos matemáticos se estima que es poco probable que la reducción en mortalidad con vacunas orales supere el 40%. Esto se debe a que la mayoría de las muertes asociadas a RVA ocurren en países de ingresos bajos y medianos, donde la efectividad de estas vacunas es menor. Este problema de desempeño en medios de bajo nivel socio-económico, se verifica también en menor efectividad sobre morbilidad y duración de la protección. En este trabajo se discuten los posible factores involucrados en este bajo rendimiento relacionados al patógeno, el huésped y factores ambientales. En Argentina, la vacuna monovalente Rotarix® (GSK) ha sido incorporada en el Calendario Nacional en 2015, sin embargo, aún no existen estudios completos de efectividad. Las observaciones iniciales de nuestro grupo registran una disminución en el número de casos de gastroenteritis aguda requi- riendo hospitalización en el Hospital de Niños Ricardo Gutiérrez (HNRG) a partir del 2015. Para los casos con diagnóstico específico de RVA la diferencia fue cada vez más significativa para los años 2016, 2017 y 2018 (p<<0,05). Comparando las medias anuales en los períodos pre y post vacunales se hallan valores de 104,7 para los años 2008-2014 y de 40,2 para los años 2015-2018 (p<0,05) lo que implica una disminución del 61,6%. También se hallaron diferencias a la baja en el número medio de internaciones por GEA por todas las causas del 19,6%.
[ABSTRACT]. By 2022, more than 100 countries had introduced one of the four vaccines authorized for global use against rotavirus; however, according to mathematical models, it is estimated that the reduction in mortality with oral vaccines is unlikely to exceed 40%. This is because the majority of RVA-associated deaths occur in low- and middle-income countries, where the effectiveness of these vaccines is lower. This performance problem in low socioeconomic environments is also verified in lower effectiveness on morbidity and duration of protection. In this work, the possible factors involved in this low performance related to the pathogen, the host and environmental factors are discussed. In Argentina, the monovalent Rotarix® (GSK) vaccine has been incorporated into the National Schedule in 2015, however, there are still no complete effectiveness studies. The initial observations of our group record a decrease in the number of cases of acute gastroenteritis requiring hospitalization at the Ricardo Gutiérrez Children's Hospital (HNRG) starting in 2015. For cases with a specific diagnosis of RVA, the difference was increasingly significant for the years 2016, 2017 and 2018 (p<<0.05). Comparing the annual averages in the pre- and post-vac- cine periods, values of 104.7 are found for the years 2008-2014 and 40.2 for the years 2015-2018 (p<0.05), which implies a decrease of 61. 6%. Downward differences were also found in the average number of hospitalizations for AGE due to all causes of 19.6%.
Asunto(s)
Infecciones por Rotavirus , Rotavirus , Vacunas contra Rotavirus , Eficacia de las Vacunas , Vulnerabilidad SocialRESUMEN
Group A rotaviruses (RVA) are the most frequent etiological agents causing severe diarrhea in infants and surveillance of genotype, and genetic characteristics of circulating strains are necessary in order to evaluate vaccine programs. The objectives of this work were to describe G and P genotype from 2012 through 2014 in Buenos Aires, Argentina completing an overview of 19 years of genotype surveillance in our region and to characterize an emerging G1P[8] strain associated with severe cases and five fatalities in 2014. We performed genotyping by RT-PCR. The sequencing of several genes, phylogenetic analyses, and comparative epidemiological data were used to know the origin and phylogenetic relationships of the emerging G1P[8] strain. Along with this report, 19 years of continuous RVA genotype surveillance in Argentina in the pre-vaccine era was covered. During the last year of this surveillance, 2014, a significantly increased incidence of RVA associated gastroenteritis was related to the reemergence of G1P[8] strains, being these ones detected in low frequency in the last nine years. Interestingly, the patients affected were significantly older when compared with those from the last six seasons. Additionally, phylogenetic analysis of several genes infer that these G1P[8] strains were closely related to Asian strains circulating during 2012 and 2013. In addition to this, the suggested extra continental origin for the 2014 G1P[8] strains and the very low circulation of G1 type during nine years probably explain the increased incidence and severity in the gastroenteritis cases and the particular epidemiologic characteristics. In conclusion, this work gives us a whole panorama of the pre-vaccine era of the RVA molecular epidemiology in the most populated region of Argentina. In this way, this work inspires us to continue with this type of studies in the post-vaccination era.
Asunto(s)
Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/virología , Infecciones por Rotavirus/epidemiología , Infecciones por Rotavirus/virología , Rotavirus/clasificación , Argentina/epidemiología , Proteínas de la Cápside/genética , Enfermedades Transmisibles Emergentes/historia , Genoma Viral , Genotipo , Historia del Siglo XXI , Humanos , Incidencia , Epidemiología Molecular , Filogenia , Prevalencia , Vigilancia en Salud Pública , ARN Viral , Rotavirus/inmunología , Infecciones por Rotavirus/historia , Infecciones por Rotavirus/prevención & control , Vacunas contra RotavirusRESUMEN
Rotaviruses are the primary cause of acute gastroenteritis in children worldwide. Although the implementation of live attenuated vaccines has reduced the number of rotavirus-associated deaths, variance in their effectiveness has been reported in different countries. This fact, among other concerns, leads to continuous efforts for the development of new generation of vaccines against rotavirus.In this work, we describe the obtention of cell wall-derived particles from a recombinant Lactococcus lactis expressing a cell wall-anchored version of the rotavirus VP6 protein. After confirming by SDS-PAGE, Western blot, flow cytometry and electronic immunomicroscopy that these particles were carrying the VP6 protein, their immunogenic potential was evaluated in adult BALB/c mice. For that, mucosal immunizations (oral or intranasal), with or without the dmLT [(double mutant Escherichia coli heat labile toxin LT(R192G/L211A)] adjuvant were performed. The results showed that these cell wall-derived particles were able to generate anti-rotavirus IgG and IgA antibodies only when administered intranasally, whether the adjuvant was present or not. However, the presence of dmLT was necessary to confer protection against rotavirus infection, which was evidenced by a 79.5 percent viral shedding reduction.In summary, this work describes the production of cell wall-derived particles which were able to induce a protective immune response after intranasal immunization. Further studies are needed to characterize the immune response elicited by these particles as well as to determine their potential as an alternative to the use of live L. lactis for mucosal antigen delivery.
Asunto(s)
Antígenos Virales/inmunología , Proteínas de la Cápside/inmunología , Pared Celular/metabolismo , Portadores de Fármacos/metabolismo , Lactococcus lactis/citología , Membrana Mucosa/metabolismo , Infecciones por Rotavirus/prevención & control , Rotavirus/fisiología , Animales , Especificidad de Anticuerpos , Antígenos Virales/metabolismo , Proteínas de la Cápside/metabolismo , Modelos Animales de Enfermedad , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
Group C Rotavirus (RVC) has been associated globally with sporadic outbreaks of gastroenteritis in children and adults. RVC also infects animals, and interspecies transmission has been reported as well as its zoonotic potential. Considering its genetic diversity and the absence of effective vaccines, it is important and necessary to develop new generation vaccines against RVC for both humans and animals. The aim of the present study was to develop and characterize an HSV-1-based amplicon vector expressing a human RVC-VP6 protein and evaluate the humoral immune response induced after immunizing BALB/c mice. Local fecal samples positive for RVC were used for isolation and sequencing of the vp6 gene, which phylogenetically belongs to the I2 genotype. We show here that cells infected with the HSV[VP6C] amplicon vector efficiently express the VP6 protein, and induced specific anti-RVC antibodies in mice immunized with HSV[VP6C], in a prime-boost schedule. This work highlights that amplicon vectors are an attractive platform for the generation of safe genetic immunogens against RVC, without the addition of external adjuvants.
Asunto(s)
Antígenos Virales/genética , Antígenos Virales/inmunología , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Expresión Génica , Vectores Genéticos/genética , Herpesvirus Humano 1/genética , Rotavirus/genética , Rotavirus/inmunología , Animales , Anticuerpos Antivirales/inmunología , Chlorocebus aethiops , Humanos , Inmunidad Humoral , Masculino , Ratones , Filogenia , Proteínas Recombinantes , Células VeroRESUMEN
BACKGROUND: Group A rotaviruses (RVA) are the most frequent single etiological agents of severe diarrhea in infants. Since 2006 RVA vaccines have been introduced in national schedules of middle and high income countries with substantial declines in rotavirus associated disease burden. However, surveillance must be maintained to, eventually, detect emerging types or variants selected by the new pressure imposed by vaccination. OBJECTIVES: To analyze the molecular epidemiology of group A rotavirus after vaccine introduction in the region in the context of data from more than 15 years of continuous surveillance in Buenos Aires. STUDY DESIGN: RVA positive diarrhea samples collected in Buenos Aires from 2008 to 2011 were genotyped by RT-PCR. Selected samples were sequenced to gain insight on evolution of common and globally emerging human RVA strains. RESULTS: Lineage III G12P[8] strain emerged in 2008 in Buenos Aires and shared co-dominancy with G3 strains during 2009. An atypical long lasting circulation of G2P[4] strains since 2004 reached rates around 80% in 2011 in Buenos Aires. Sequencing of the VP7 and VP4 genes of representative G2P[4] isolates suggests Brazil as the origin of the 2010-2011 strains. CONCLUSIONS: Globally emergent G12 lineage III strains could be established as dominant strains in a very populated area in two years since emergence. In this work it was also shown that the persistence of G2P[4] strains during 8 years could be related to massive immunization with the monovalent vaccine in the region.
Asunto(s)
Infecciones por Rotavirus/epidemiología , Infecciones por Rotavirus/prevención & control , Vacunas contra Rotavirus/inmunología , Rotavirus/aislamiento & purificación , Brasil/epidemiología , Proteínas de la Cápside/genética , Heces/virología , Genotipo , Historia del Siglo XXI , Humanos , Vacunación Masiva , Datos de Secuencia Molecular , Filogenia , Vigilancia en Salud Pública , ARN Viral/genética , Rotavirus/clasificación , Rotavirus/genética , Infecciones por Rotavirus/historia , Infecciones por Rotavirus/virologíaRESUMEN
Group A rotaviruses (RV-A) are the most common agents of viral gastroenteritis in children worldwide. The goal of this study was to compare two different methods to concentrate RV-A from sewage samples and to improve the detection and quantification of RV-A using a multiplex quantitative PCR assay with an internal control. Both RV-A and the internal control virus, bacteriophage PP7, were seeded into wastewater and then concentrated using either an ultrafiltration-based adsorption-elution protocol or an ultracentrifugation-based protocol. Real time multiplex quantitative PCR was used to quantify the purified RV-A and PP7, and the results of the multiplex assay were compared with the results of the monoplex assays. The ultracentrifugation-based method had a mean recovery rate of 47% (range: 34-60%), while the ultrafiltration-based adsorption-elution method had a mean recovery rate of 3.5% (range: 1.5-5.5%). These results demonstrate that ultracentrifugation is a more appropriate method for recovering RV-A from wastewater. This method together with the multiplex qPCR assay may be suitable for routine laboratory use.
Asunto(s)
Reacción en Cadena de la Polimerasa , ARN Viral/aislamiento & purificación , Rotavirus/aislamiento & purificación , Aguas del Alcantarillado/virología , Ultracentrifugación/métodos , Adsorción , Gastroenteritis/virología , Humanos , Levivirus , Rotavirus/genética , Ultrafiltración/métodos , Microbiología del AguaRESUMEN
Detection and characterization of group A rotavirus in Buenos Aires, Argentina, was conducted on 710 fecal samples from children 0-15 years old collected between 2004 and 2007. Rotavirus was detected in 140 (19.7%) samples with G9P[8] (30.0%) and G2P[4] (21.4%) as the most common genotypes. Mixed (G and/or P) infections accounted for 17.9% of the samples and the emerging G12 strain was detected during 2004 (3.5%) and 2007 (2.5%). Genotype G2 was the most prevalent during 2004 (43.9%) and 2007 (57.5%) and G9 during 2005 (58.0%) and 2006 (61.5%). Analysis of genotype prevalences from studies performed since 1996 in the same area showed striking natural fluctuations in G and P genotype frequencies. In particular, G2P[4] strains disappeared after 1999 and reemerged in 2004 to become the predominant strain by 2007 with a concomitant major decrease in G1P[8] prevalence. The VP7 genes from Argentinian G9 and G2 strains were sequenced and phylogenetic analysis was conducted in order to compare with sequences from strains isolated in regional countries reported previously. Several changes in the deduced amino acid sequence in antigenic regions of the VP7 protein from Argentinian and Brazilian strains were identified compared to vaccine strains. Overall, this study revealed relationships in the circulation of rotavirus strains in South American countries and major replacements in dominant genotypes, including the virtual disappearance of G1P[8] strains in a non-vaccinated population. High numbers of mixed infections speeding up evolution, circulation of rare serotypes, and antigenic drift could, eventually, become challenges for new vaccines.
Asunto(s)
Infecciones por Rotavirus/epidemiología , Infecciones por Rotavirus/virología , Rotavirus/clasificación , Rotavirus/genética , Adolescente , Antígenos Virales/genética , Argentina , Brasil/epidemiología , Proteínas de la Cápside/genética , Niño , Preescolar , Análisis por Conglomerados , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Epidemiología Molecular , Datos de Secuencia Molecular , Filogenia , ARN Viral/genética , Rotavirus/aislamiento & purificación , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
The role of group C rotaviruses as a cause of diarrhea was examined among children <17 years of age admitted to a Hospital in a suburban area of Buenos Aires, Argentina between 1997 and 2003. A total of 1,579 fecal samples were screened for group A (RVA) and C (RVC) rotaviruses by two in-house ELISA methods at Quilmes University (UNQ-ELISA). Samples positive, doubtful and negative by RVC specific UNQ-ELISA (n = 246) were examined further for RVC by another in-house ELISA (CDC-ELISA), electron microscopy, RT-PCR, nested PCR, and Southern hybridization. Sensitivity, specificity, and predictive values for each test were determined. While the sensitivity was comparable for the nested PCR and CDC-ELISA methods (82.5%), the molecular methods were slightly more specific. Poorly preserved particles were often seen in fecal samples, suggesting that degradation of RNA could be a factor influencing the performance of molecular methods. The incidence of RVC was estimated to be 3% without apparent differences among seasons. RVC infected patients had a significantly (P < 0.001) higher median age (6 years vs. 1 year) than those with RVA infection. Sequence of the RVC VP7 gene from six Argentinean strains and sequences reported previously in different countries showed high nucleotide (94.4-99.9%) sequence identities, indicating a high degree of conservation for human RVC VP7 genes among strains collected on five continents over a period of 17 years. These findings indicate that RVC is a significant cause of diarrhea and it is necessary to develop simple and sensitive serological methods for its detection.
Asunto(s)
Diarrea/virología , Infecciones por Rotavirus/diagnóstico , Rotavirus/clasificación , Rotavirus/aislamiento & purificación , Adolescente , Antígenos Virales/análisis , Argentina , Southern Blotting/métodos , Niño , Preescolar , Secuencia Conservada , Ensayo de Inmunoadsorción Enzimática/métodos , Heces/virología , Hospitales , Humanos , Lactante , Recién Nacido , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Valor Predictivo de las Pruebas , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Rotavirus/genética , Rotavirus/inmunología , Infecciones por Rotavirus/virología , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Homología de SecuenciaRESUMEN
The circulation of the unusual P[9]G12 strains was previously reported in suburban Buenos Aires, Argentina and in Far Eastern Asian countries. To examine genetic relationships of these strains the genes coding VP7, VP4, and NSP1 from two Argentine, one Japanese and one Korean P[9]G12 isolates were sequenced and their overall genome relatedness was determined by liquid hybridization. In addition, liquid hybridization was used to compare this group of strains to the previous G12 isolates L26 and Se585, and prototype Wa, DS-1, and AU-1 strains. The genomes of the Argentinean, Japanese and Korean strains were virtually indistinguishable by hybridization assays, suggesting very high sequence relatedness for all 11 segments. Hybridization assays also demonstrated that these four strains belong to the AU-1 genogroup and that their genetic relationship with rotaviruses L26 and Se585 is limited to the VP7 gene. The VP7, VP4, and NSP1 genes of the Argentinean, Japanese and Korean strains were highly homologous to each other and to Thai strain T152 ( approximately 99% identity). These results together with the report of a similar strain detected during 2003 in Brazil are consistent with a recent importation and dissemination of the G12 strains from Far Eastern countries into South America. Increasing reports from several regions of the world demonstrating a variety of different G12 reassortant strains suggests that routine surveillance for this serotype should be conducted to determine its potential for global emergence.
Asunto(s)
Infecciones por Rotavirus/virología , Rotavirus/genética , Animales , Antígenos Virales/genética , Argentina , Proteínas de la Cápside/genética , Línea Celular , Niño , Femenino , Genotipo , Humanos , Lactante , Japón , Corea (Geográfico) , Masculino , Datos de Secuencia Molecular , Filogenia , Rotavirus/clasificación , Rotavirus/aislamiento & purificación , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Proteínas no Estructurales Virales/genéticaRESUMEN
In spite of active measles virus (MV) vaccination strategies, reemergence continues to occur, impairing global eradication programs. The immune status against measles was evaluated in 350 vaccinated healthy Argentine children and teenagers who received a single dose of the MV Schwarz strain Lirugen vaccine (Aventis Pasteur). Sera were assessed for immunoglobulin G (IgG) antibodies by a commercial enzyme immunoassay (EIA) (Enzygnost; Behring), an in-house EIA, and neutralization EIA. Results obtained with these methods showed a marked decline in IgG level with increasing age. At 1 to 4 years of age, 84% of children had IgG antibodies above 200 mIU/ml, conventionally accepted as protective levels, whereas only 32% of older children and teenagers had antibody levels exceeding 200 mIU/ml. Moreover, the MV IgG content in the teenage group was significantly lower than the IgG antibody level of the group of younger children (P < 0.0001). In contrast, screening for IgG antibody levels to inactivated tetanus vaccine showed that, on average, 80% of this population was fully protected and that this high level of protection remained through the teenage years. This study suggests that within this population a considerable proportion of individuals had low measles antibody levels that may be insufficient to protect against reinfections or clinical disease.
Asunto(s)
Anticuerpos Antivirales/sangre , Vacuna Antisarampión/inmunología , Virus del Sarampión/inmunología , Sarampión/prevención & control , Adolescente , Factores de Edad , Anticuerpos Antibacterianos/sangre , Argentina , Niño , Preescolar , Femenino , Humanos , Técnicas para Inmunoenzimas , Inmunoglobulina G/sangre , Lactante , Masculino , Vacuna Antisarampión/administración & dosificación , Pruebas de Neutralización , Estadística como Asunto , Toxoide Tetánico/inmunologíaRESUMEN
The incidence of human group C rotavirus infections among children and adults in Buenos Aires was evaluated by enzyme linked immunosorbent assays (ELISA) based on recombinant group C VP6 protein (Cowden strain). A total of 976 stool samples taken from patients (ages 6 months to 15 years) with acute diarrhea were tested for the presence of group C rotavirus. Among these, only 10 (1.02%) were group C rotavirus positive by enzyme-linked immunosorbent assay (ELISA) confirmed by absorption with group C VP6 antibodies and by RT-PCR for both VP6 and VP7 genes. The average age (5.86 years) was significantly superior to that in group A-infected patients (1.63 years). Previous exposure to this virus was assessed by detecting specific IgG in sera taken from healthy individuals grouped by age. Of 844 sera tested, 425 (50.3%) were group C IgG positive by ELISA, confirmed by Western blot analysis. The rates of IgG positivity for group A and C rotaviruses during the first years of life indicated that infections with group C are frequent in older children (3-5 years), whereas group A infections are prevalent in infants and young children (6-18 months). This study shows that group C rotavirus infections in Argentine children occur later in life than group A and are relatively common in spite of the low detection rate of this virus.