Virulence Profiles of Wild-Type, P.1 and Delta SARS-CoV-2 Variants in K18-hACE2 Transgenic Mice.

Since December 2019, the world has been experiencing the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and we now face the emergence of several variants. We aimed to assess the differences between the wild-type (Wt) (Wuhan) strain and the P.1 (Gamma) and Delta variants using infected K18-hACE2 mice. The clinical manifestations, behavior, virus load, pulmonary capacity, and histopathological alterations were analyzed. The P.1-infected mice showed weight loss and more severe clinical manifestations of COVID-19 than the Wt and Delta-infected mice. The respiratory capacity was reduced in the P.1-infected mice compared to the other groups. Pulmonary histological findings demonstrated that a more aggressive disease was generated by the P.1 and Delta variants compared to the Wt strain of the virus. The quantification of the SARS-CoV-2 viral copies varied greatly among the infected mice although it was higher in P.1-infected mice on the day of death. Our data revealed that K18-hACE2 mice infected with the P.1 variant develop a more severe infectious disease than those infected with the other variants, despite the significant heterogeneity among the mice.

Virulence profiles of wild-type, P.1 and delta SARS-CoV-2 variants in K18-hACE2 transgenic mice

Since December 2019, the world has been experiencing the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and we now face the emergence of several variants. We aimed to assess the differences between the wild-type (Wt) (Wuhan) strain and the P.1 (Gamma) and Delta variants using infected K18-hACE2 mice. The clinical manifestations, behavior, virus load, pulmonary capacity, and histopathological alterations were analyzed. The P.1-infected mice showed weight loss and more severe clinical manifestations of COVID-19 than the Wt and Delta-infected mice. The respiratory capacity was reduced in the P.1-infected mice compared to the other groups. Pulmonary histological findings demonstrated that a more aggressive disease was generated by the P.1 and Delta variants compared to the Wt strain of the virus. The quantification of the SARS-CoV-2 viral copies varied greatly among the infected mice although it was higher in P.1-infected mice on the day of death. Our data revealed that K18-hACE2 mice infected with the P.1 variant develop a more severe infectious disease than those infected with the other variants, despite the significant heterogeneity among the mice.

Mutational profile confers increased stability of SARS-CoV-2 spike protein in Brazilian isolates.

Spike (S) protein has been recognized as a promising molecular target for diagnostic, vaccines and antiviral drugs development for COVID-19. In this study, we analyzed the most predominant mutations in the S protein of Brazilian isolates and predicted the effect of these amino acid alterations to protein conformation. A total of 25,924 sequences were obtained from GISAID for five regions of Brazilian territory (Midwest, North, Northeast, South, and Southeast), according to exclusion criteria. Most of the SARS-CoV-2 isolates belongs to the G clade and showed a large occurrence of D614G, N501Y and L18F substitutions. Prediction effects of these amino acid substitutions on the structure dynamics of the spike protein indicated a positive ΔΔG values and negative ΔΔSVib in most cases which is associated to structural stabilization and flexibility reduction of the S protein. Mutations E484K, N501Y and K417N belong to several SARS-CoV-2 variants of concern such as Alpha, Beta, Gamma and Delta, and showed high incidence among Brazilian isolates. These mutations have been described to increase RBD affinity to ACE-2 host and abolishment of RBD affinity to potent neutralizing ant-RBD. The increase in rates of infection and reinfection requires continuous genomic surveillance studies in order to characterize emerging mutations and monitor vaccine efficacy, and thus consideration structural data and dynamics in the observed phenotypes.Communicated by Ramaswamy H. Sarma.

Development of a new DNA vaccine based on mycobacterial ESAT-6 antigen delivered by recombinant invasive Lactococcus lactis FnBPA+.

The use of the food-grade bacterium Lactococcus lactis as a vehicle for the oral delivery of DNA vaccine plasmids constitutes a promising strategy for vaccination. The delivery of DNA plasmids into eukaryotic cells is of critical importance for subsequent DNA expression and effectiveness of the vaccine. In this context, the use of the recombinant invasive L. lactis FnBPA+ (fibronectin-binding protein A) strain for the oral delivery of the eukaryotic expression vector vaccination using lactic acid bacteria (pValac), coding for the 6-kDa early secreted antigenic target (ESAT-6) gene of Mycobacterium tuberculosis, could represent a new DNA vaccine strategy against tuberculosis. To this end, the ESAT-6 sequence was cloned into the pValac vector; the L. lactis fibronectin-binding protein A (FnBPA)+ (pValac:ESAT-6) strain was obtained, and its immunological profile was checked in BALB/c mice. This strain was able to significantly increase interferon gamma (IFN-γ) production in spleen cells, showing a systemic T helper 1 (Th1) cell response. The mice also showed a significant increase in specific secretory immunoglobulin A (sIgA) production in colon tissue and fecal extracts. Thus, this is the first time that L. lactis has been used to deliver a plasmid DNA harboring a gene that encodes an antigen against tuberculosis through mucous membranes.

Recombinant Lactococcus lactis fails to secrete bovine chymosine.

Bovine chymosin is an important milk-clotting agent used in the manufacturing of cheeses. Currently, the production of recombinant proteins by genetically modified organisms is widespread, leading to greatly reduced costs. Lactococcus (L.) lactis, the model lactic acid bacterium, was considered a good candidate for heterologous chymosin production for the following reasons: (1) it is considered to be a GRAS (generally regarded as safe) microorganism, (2) only one protease is present on its surface, (3) it can secrete proteins of different sizes, and (4) it allows for the direct production of protein in fermented food products. Thus, three genetically modified L. lactis strains were constructed to produce and target the three different forms of bovine chymosin, prochymosin B, chymosin A and chymosin B to the extracellular medium. Although all three proteins were stably produced in L. lactis, none of the forms were detected in the extracellular medium or showed clotting activity in milk. Our hypothesis is that this secretion deficiency and lack of clotting activity can be explained by the recombinant protein being attached to the cell envelope. Thus, the development of other strategies is necessary to achieve both production and targeting of chymosin in L. lactis, which could facilitate the downstream processing and recovery of this industrially important protein.

Lactococcus lactis as a live vector: heterologous protein production and DNA delivery systems.

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as mucosal delivery vehicles for vaccinal, medical or technological use has been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins and for plasmid DNA delivery to eukaryotic cells. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium) and more recently to efficiently transfer DNA to eukaryotic cells. A promising application of L. lactis is its use for the development of live mucosal vaccines. Here, we have reviewed the expression of heterologous protein and the various delivery systems developed for L. lactis, as well as its use as an oral vaccine carrier.

Phenotypic and genetic analysis of Enterobacter spp. from a Brazilian oligotrophic freshwater lake.

We characterized a population of Enterobacter spp. of the Enterobacter cloacae complex isolated from an oligotrophic lake; most isolates were identified as E. cloacae. Fingerprinting polymerase chain reaction (PCR), along with morphological, biochemical, physiological, and plasmid profiles analyses, including antimicrobial susceptibility testing, were performed on 22 environmental isolates. Misidentification occurred when using the API 20E identification system. Analysis of 16S rDNA sequences confirmed the close relatedness between species of the E. cloacae complex. The tDNA PCR allowed the differentiation and identification of the E. cloacae isolates. Evaluation of genetic diversity by 16S rDNA sequence, tDNA, internal transcribed spacers, and enterobacterial repetitive intergenic concensus profiles revealed nearly identical isolates, although they exhibited different physiological and antimicrobial resistance profiles. Among the Enterobacter isolates, 96% were resistant to at least one antimicrobial; multiple resistance was also found at a high frequency (86%). The antimicrobials against which resistance was found most frequently were beta-lactams, chloramphenicol, and streptomycin. Plasmids were found in 21 of the 22 Enterobacter isolates. This confirms the conception that antibiotic resistance can occur in oligotrophic freshwater lake bacteria, which has important implications for public health.