Display of FliC131 on the Surface of Lactococcus lactis as a Strategy to Increase its Adjuvanticity for Mucosal Immunization.

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.

Rotavirus del grupo A circulantes en el Gran Buenos Aires durante los períodos pre- y post-vacunales / Vaccines against rotavirus and impact of their introduction in Argentina

[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%.

Pre-vaccine rotavirus surveillance in Buenos Aires, Argentina. Characterization of an emergent G1P[8] strain associated to fatal cases in 2014.

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.

Rotavirus VP6 protein mucosally delivered by cell wall-derived particles from Lactococcus lactis induces protection against infection in a murine model.

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.

Evaluation of the immunogenicity of a recombinant HSV-1 vector expressing human group C rotavirus VP6 protein.

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.

Surveillance of group A Rotavirus in Buenos Aires 2008-2011, long lasting circulation of G2P[4] strains possibly linked to massive monovalent vaccination in the region.

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.