Compensatory evolution in NusG improves fitness of drug-resistant M. tuberculosis.

Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors1-8. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy9,10. However, RifR Mtb accounts for one-quarter of all deaths due to drug-resistant bacteria11,12. We took a comparative functional genomics approach to define processes that are differentially vulnerable to CRISPR interference (CRISPRi) inhibition in RifR Mtb. Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA13. We find this pro-pausing NusG-RNAP interface to be under positive selection in clinical RifR Mtb isolates. Mutations in the NusG-RNAP interface reduce pro-pausing activity and increase fitness of RifR Mtb. Collectively, these results define excessive RNAP pausing as a molecular mechanism that drives the fitness cost of RifR in Mtb, identify a new mechanism of compensation to overcome this cost, suggest rational approaches to exacerbate the fitness cost, and, more broadly, could inform new therapeutic approaches to develop drug combinations to slow the evolution of RifR in Mtb.

Draft Genome Sequence of an Isolate of Genotype VII Newcastle Disease Virus Isolated from an Outbreak in Fighting Cock in Peru.

This study presents a draft genome sequence of a Newcastle disease virus (NDV) strain (VFAR-136) isolated from a fighting cock (Gallus gallus) in the south of Peru. Strain VFAR-136 is a new report of NDV genotype VII circulating in Peru.

Squalene in oil-based adjuvant improves the immunogenicity of SARS-CoV-2 RBD and confirms safety in animal models.

COVID-19 pandemic has accelerated the development of vaccines against its etiologic agent, SARS-CoV-2. However, the emergence of new variants of the virus lead to the generation of new alternatives to improve the current sub-unit vaccines in development. In the present report, the immunogenicity of the Spike RBD of SARS-CoV-2 formulated with an oil-in-water emulsion and a water-in-oil emulsion with squalene was evaluated in mice and hamsters. The RBD protein was expressed in insect cells and purified by chromatography until >95% purity. The protein was shown to have the appropriate folding as determined by ELISA and flow cytometry binding assays to its receptor, as well as by its detection by hamster immune anti-S1 sera under non-reducing conditions. In immunization assays, although the cellular immune response elicited by both adjuvants were similar, the formulation based in water-in-oil emulsion and squalene generated an earlier humoral response as determined by ELISA. Similarly, this formulation was able to stimulate neutralizing antibodies in hamsters. The vaccine candidate was shown to be safe, as demonstrated by the histopathological analysis in lungs, liver and kidney. These results have shown the potential of this formulation vaccine to be evaluated in a challenge against SARS-CoV-2 and determine its ability to confer protection.

Preclinical Assessment of IgY Antibodies Against Recombinant SARS-CoV-2 RBD Protein for Prophylaxis and Post-Infection Treatment of COVID-19.

Within the framework of the current COVID-19 pandemic, there is a race against time to find therapies for the outbreak to be controlled. Since vaccines are still tedious to develop and partially available for low-income countries, passive immunity based on egg-yolk antibodies (IgY) is presented as a suitable approach to preclude potential death of infected patients, based on its high specificity/avidity/production yield, cost-effective manufacture, and ease of administration. In the present study, IgY antibodies against a recombinant RBD protein of SARS-CoV-2 were produced in specific-pathogen-free chickens and purified from eggs using a biocompatible method. In vitro immunoreactivity was tested, finding high recognition and neutralization values. Safety was also demonstrated prior to efficacy evaluation, in which body weight, kinematics, and histopathological assessments of hamsters challenged with SARS-CoV-2 were performed, showing a protective effect administering IgY intranasally both as a prophylactic treatment or a post-infection treatment. The results of this study showed that intranasally delivered IgY has the potential to both aid in prevention and in overcoming COVID-19 infection, which should be very useful to control the advance of the current pandemic and the associated mortality.

Intranasal vaccination of hamsters with a Newcastle disease virus vector expressing the S1 subunit protects animals against SARS-CoV-2 disease.

The coronavirus disease-19 (COVID-19) pandemic has already claimed millions of lives and remains one of the major catastrophes in the recorded history. While mitigation and control strategies provide short term solutions, vaccines play critical roles in long term control of the disease. Recent emergence of potentially vaccine-resistant and novel variants necessitated testing and deployment of novel technologies that are safe, effective, stable, easy to administer, and inexpensive to produce. Here we developed three recombinant Newcastle disease virus (rNDV) vectored vaccines and assessed their immunogenicity, safety, and protective efficacy against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in mice and hamsters. Intranasal administration of rNDV-based vaccine candidates elicited high levels of neutralizing antibodies. Importantly, the nasally administrated vaccine prevented lung damage, and significantly reduced viral load in the respiratory tract of vaccinated animal which was compounded by profound humoral immune responses. Taken together, the presented NDV-based vaccine candidates fully protected animals against SARS-CoV-2 challenge and warrants evaluation in a Phase I human clinical trial as a promising tool in the fight against COVID-19.

A novel enolase from Taenia solium metacestodes and its evaluation as an immunodiagnostic antigen for porcine cysticercosis.

Cysticercosis is a worldwide parasitic disease of humans and pigs principally caused by infection with the larvae of the pork tapeworm Taenia solium. Through the use of the recently-made-available T. solium genome, we identified a gene within a novel 1448 bp ORF that theoretically encodes for a 433 amino acid-long protein and predicted to be an α-enolase closely related to enolases of other flatworms. Additional bioinformatic analyses revealed a putative plasminogen-binding region on this protein, suggesting a potential role for this protein in pathogenesis. On this basis, we isolated the mRNA encoding for this presumptive enolase from T. solium metacestodes and reverse-transcribed it into cDNA before subsequently cloning and expressing it in both E. coli (rEnoTs) and insect cells (rEnoTsBac), in a 6xHis tagged manner. The molecular weights of these two recombinant proteins were ∼48 and ∼50 kDa, respectively, with the differences likely attributable to differential glycosylation. We used spectrophotometric assays to confirm the enolase nature of rEnoTs as well as to measure its enzymatic activity. The resulting estimates of specific activity (60.000 U/mg) and Km (0.091 mM) are quite similar to the catalytic characteristics of enolases of other flatworms. rEnoTs also exhibited high immunogenicity, eliciting a strong polyclonal antibody response in immunized rabbits. We subsequently employed rEnoTsBac for use in an ELISA aimed at discriminating between healthy pigs and those infected with T. solium. This diagnostic assay exhibited a sensitivity of 88.4% (95% CI, 74.92%-96.11%) and a specificity of 83.7% (95% CI: 69.29%-93.19%). In conclusión, this study reports on and enzymatically characterizes a novel enolase from T. solium metacestode, and shows a potential use as an immunodiagnostic for porcine cysticercosis.

Genetic variability of Taenia solium cysticerci recovered from experimentally infected pigs and from naturally infected pigs using microsatellite markers.

The adult Taenia solium, the pork tapeworm, usually lives as a single worm in the small intestine of humans, its only known definitive host. Mechanisms of genetic variation in T. solium are poorly understood. Using three microsatellite markers previously reported [1], this study explored the genetic variability of T. solium from cysts recovered from experimentally infected pigs. It then explored the genetic epidemiology and transmission in naturally infected pigs and adult tapeworms recovered from human carriers from an endemic rural community in Peru. In an initial study on experimental infection, two groups of three piglets were each infected with proglottids from one of two genetically different tapeworms for each of the microsatellites. After 7 weeks, pigs were slaughtered and necropsy performed. Thirty-six (92.3%) out of 39 cysts originated from one tapeworm, and 27 (100%) out of 27 cysts from the other had exactly the same genotype as the parental tapeworm. This suggests that the microsatellite markers may be a useful tool for studying the transmission of T. solium. In the second study, we analyzed the genetic variation of T. solium in cysts recovered from eight naturally infected pigs, and from adult tapeworms recovered from four human carriers; they showed genetic variability. Four pigs had cysts with only one genotype, and four pigs had cysts with two different genotypes, suggesting that multiple infections of genetically distinct parental tapeworms are possible. Six pigs harbored cysts with a genotype corresponding to one of the identified tapeworms from the human carriers. In the dendrogram, cysts appeared to cluster within the corresponding pigs as well as with the geographical origin, but this association was not statistically significant. We conclude that genotyping of microsatellite size polymorphisms is a potentially important tool to trace the spread of infection and pinpoint sources of infection as pigs spread cysts with a shared parental genotype.