ABSTRACT
BACKGROUND: Rift Valley fever virus, a pathogen to ruminants, camelids, and humans, is an emerging mosquito-borne bunyavirus currently endemic to Africa and the Arabian Peninsula. Although animals are primarily infected via mosquito bites, humans mainly become infected following contact with infected tissues or fluids of infected animals. There is an urgent need for adequate countermeasures, especially for humans, because effective therapeutics or vaccines are not yet available. Here we assessed the safety, tolerability, and immunogenicity of a next-generation, four-segmented, live-attenuated vaccine candidate, referred to as hRVFV-4s, in humans. METHODS: A first-in-human, single-centre, randomised, double-blind, placebo-controlled trial was done in Belgium in which a single dose of hRVFV-4s was administered to healthy volunteers aged 18-45 years. Participants were randomly assigned using an interactive web response system. The study population encompassed 75 participants naive to Rift Valley fever virus infection, divided over three dosage groups (cohorts) of 25 participants each. All participants were followed up until 6 months. Using a staggered dose escalating approach, 20 individuals of each cohort were injected in the deltoid muscle of the non-dominant arm with either 104 (low dose), 105 (medium dose), or 106 (high dose) of 50% tissue culture infectious dose of hRVFV-4s as based on animal data, and five individuals per cohort received formulation buffer as a placebo. Primary outcome measures in the intention-to-treat population were adverse events and tolerability. Secondary outcome measures were vaccine-induced viraemia, vaccine virus shedding, Rift Valley fever virus nucleocapsid antibody responses (with ELISA), and neutralising antibody titres. Furthermore, exploratory objectives included the assessment of cellular immune responses by ELISpot. The trial was registered with the EU Clinical Trials Register, 2022-501460-17-00. FINDINGS: Between August and December, 2022, all 75 participants were vaccinated. No serious adverse events or vaccine-related severe adverse events were reported. Pain at the injection site (51 [85%] of 60 participants) was most frequently reported as solicited local adverse event, and headache (28 [47%] of 60) and fatigue (28 [47%] of 60) as solicited systemic adverse events in the active group. No vaccine virus RNA was detected in any of the blood, saliva, urine, or semen samples. Rift Valley fever virus nucleocapsid antibody responses were detected in most participants who were vaccinated with hRVFV-4s (43 [72%] of 60 on day 14) irrespective of the administered dose. In contrast, a clear dose-response relationship was observed for neutralising antibodies on day 28 with four (20%) of 20 participants responding in the low-dose group, 13 (65%) of 20 responding in the medium-dose group, and all participants (20 [100%] of 20) responding in the high-dose group. Consistent with the antibody responses, cellular immune responses against the nucleocapsid protein were detected in all dose groups, whereas a more dose-dependent response was observed for the Gn and Gc surface glycoproteins. Neutralising antibody titres declined over time, whereas nucleocapsid antibody responses remained relatively stable for at least 6 months. INTERPRETATION: The hRVFV-4s vaccine showed a high safety profile and excellent tolerability across all tested dose regimens, eliciting robust immune responses, particularly with the high-dose administration. The findings strongly support further clinical development of this candidate vaccine for human use. FUNDING: The Coalition for Epidemic Preparedness Innovations with support from the EU Horizon 2020 programme.
ABSTRACT
Panels of pre- and post-pandemic farm animals, wild boar and human sera, including human sera able to neutralize SARS-CoV-2 in vitro, were tested in serological tests to determine their cross-reactivity with ß- and α-CoV originating from farm animals. Sera were tested in neutralization assays with high ascending concentrations (up to 1 × 104 TCID50 units/well) of ß-CoV Bovine coronavirus (BCV), SARS-CoV-2, and porcine α-CoV-transmissible gastroenteritis virus (TGEV). In addition, sera were tested for immunostaining of cells infected with ß-CoV porcine hemagglutinating encephalomyelitis (PHEV). Testing revealed a significantly higher percentage of BCV neutralization (78%) for sera of humans that had experienced a SARS-CoV-2 infection (SARS-CoV-2 convalescent sera) than was observed for human pre-pandemic sera (37%). Also, 46% of these human SARS-CoV-2 convalescent sera neutralized the highest concentration of BCV (5 × 103 TCID50/well) tested, whereas only 9.6% of the pre-pandemic sera did. Largely similar percentages were observed for staining of PHEV-infected cells by these panels of human sera. Furthermore, post-pandemic sera collected from wild boars living near a densely populated area in The Netherlands also showed a higher percentage (43%) and stronger BCV neutralization than was observed for pre-pandemic sera from this area (21%) and for pre- (28%) and post-pandemic (20%) sera collected from wild boars living in a nature reserve park with limited access for the public. High percentages of BCV neutralization were observed for pre- and post-pandemic sera of cows (100%), pigs (up to 45%), sheep (36%) and rabbits (60%). However, this cross-neutralization was restricted to sera collected from specific herds or farms. TGEV was neutralized only by sera of pigs (68%) and a few wild boar sera (4.6%). None of the BCV and PHEV cross-reacting human pre-pandemic, wild boar and farm animal sera effectively neutralized SARS-CoV-2 in vitro. Preexisting antibodies in human sera effectively neutralized the animal ß-CoV BCV in vitro. This cross-neutralization was boosted after humans had experienced a SARS-CoV-2 infection, indicating that SARS-CoV-2 activated a "memory" antibody response against structurally related epitopes expressed on the surface of a broad range of heterologous CoV, including ß-CoV isolated from farm animals. Further research is needed to elucidate if a symptomless infection or environmental exposure to SARS-CoV-2 or another ß-CoV also triggers such a "memory" antibody response in wild boars and other free-living animals.
Subject(s)
COVID-19 , Transmissible gastroenteritis virus , Humans , Female , Animals , Cattle , Rabbits , Sheep , Swine , Animals, Domestic , SARS-CoV-2 , Pandemics , COVID-19/epidemiology , COVID-19/veterinary , COVID-19 Serotherapy , Sus scrofaABSTRACT
In assessing species susceptibility for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and in the search for an appropriate animal model, multiple research groups around the world inoculated a broad range of animal species using various SARS-CoV-2 strains, doses and administration routes. Although in silico analyses based on receptor binding and diverse in vitro cell cultures were valuable, exact prediction of species susceptibility based on these tools proved challenging. Here, we assessed whether precision-cut lung slices (PCLS) could facilitate the selection of animal models, thereby reducing animal experimentation. Pig, hamster and cat PCLS were incubated with SARS-CoV-2 and virus replication was followed over time. Virus replicated efficiently in PCLS from hamsters and cats, while no evidence of replication was obtained for pig PCLS. These data corroborate the findings of many research groups that have investigated the susceptibility of hamsters, pigs and cats towards infection with SARS-CoV-2. Our findings suggest that PCLS can be used as convenient tool for the screening of different animal species for sensitivity to newly emerged viruses. To validate our results obtained in PCLS, we employed the hamster model. Hamsters were inoculated with SARS-CoV-2 via the intranasal route. Susceptibility to infection was evaluated by body weight loss, viral loads in oropharyngeal swabs and respiratory tissues and lung pathology. The broadly used hamster model was further refined by including activity tracking of the hamsters by an activity wheel as a very robust and sensitive parameter for clinical health. In addition, to facilitate the quantification of pathology in the lungs, we devised a semi-quantitative scoring system for evaluating the degree of histological changes in the lungs. The inclusion of these additional parameters refined and enriched the hamster model, allowing for the generation of more data from a single experiment.
ABSTRACT
Streptococcus suis is a Gram-positive bacterium and zoonotic pathogen that causes meningitis and sepsis in pigs and humans. The aim of this study was to identify genes required for S. suis infection. We created Tn-Seq libraries in a virulent S. suis strain 10, which was used to inoculate pigs in an intrathecal experimental infection. Comparative analysis of the relative abundance of mutants recovered from different sites of infection (blood, cerebrospinal fluid, and meninges of the brain) identified 361 conditionally essential genes, i.e. required for infection, which is about 18% of the genome. The conditionally essential genes were primarily involved in metabolic and transport processes, regulation, ribosomal structure and biogenesis, transcription, and cell wall membrane and envelope biogenesis, stress defenses, and immune evasion. Directed mutants were created in a set of 10 genes of different genetic ontologies and their role was determined in ex vivo models. Mutants showed different levels of sensitivity to survival in whole blood, serum, cerebrospinal fluid, thermic shock, and stress conditions, as compared to the wild type. Additionally, the role of three selected mutants was validated in co-infection experiments in which pigs were infected with both wild type and isogenic mutant strains. The genetic determinants of infection identified in this work contribute to novel insights in S. suis pathogenesis and could serve as targets for novel vaccines or antimicrobial drugs.
Subject(s)
Genes, Bacterial , Genes, Essential , Streptococcal Infections/veterinary , Streptococcus suis/genetics , Swine Diseases/microbiology , Animals , Coinfection/microbiology , Disease Models, Animal , Immune Evasion , Meningitis , Mutation , Streptococcal Infections/microbiology , Streptococcus suis/pathogenicity , Swine , VirulenceABSTRACT
Streptococcus suis is a Gram-positive bacterium and a zoonotic pathogen residing in the nasopharynx or the gastrointestinal tract of pigs with a potential of causing life-threatening invasive disease. It is endemic in the porcine production industry worldwide, and it is also an emerging human pathogen. After invasion, the pathogen adapts to cause bacteremia and disseminates to different organs including the brain. To gain insights in this process, we infected piglets with a highly virulent strain of S. suis, and bacterial transcriptomes were obtained from blood and different organs (brain, joints, and heart) when animals had severe clinical symptoms of infection. Microarrays were used to determine the genome-wide transcriptional profile at different infection sites and during growth in standard growth medium in vitro. We observed differential expression of around 30% of the Open Reading Frames (ORFs) and infection-site specific patterns of gene expression. Genes with major changes in expression were involved in transcriptional regulation, metabolism, nutrient acquisition, stress defenses, and virulence, amongst others, and results were confirmed for a subset of selected genes using RT-qPCR. Mutants were generated in two selected genes, and the encoded proteins, i.e., NADH oxidase and MetQ, were shown to be important virulence factors in coinfection experiments and in vitro assays. The knowledge derived from this study regarding S. suis gene expression in vivo and identification of virulence factors is important for the development of novel diagnostic and therapeutic strategies to control S. suis disease.
Subject(s)
Adaptation, Physiological/genetics , Streptococcal Infections/veterinary , Streptococcus suis/genetics , Streptococcus suis/pathogenicity , Transcriptome , Virulence Factors/genetics , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Microarray Analysis , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Mutation , NADH, NADPH Oxidoreductases/genetics , NADH, NADPH Oxidoreductases/metabolism , Streptococcal Infections/microbiology , Swine , Swine Diseases/microbiology , Virulence Factors/metabolismABSTRACT
BACKGROUND: Streptococcus pneumoniae, a Gram-positive bacterium carried in the human nasopharynx, is an important human pathogen causing mild diseases such as otitis media and sinusitis as well as severe diseases including pneumonia, meningitis and sepsis. There is a strong resemblance between the anatomy, immunology and physiology of the pig and human species. Furthermore, there are striking similarities between S. suis pathogenesis in piglets and S. pneumoniae pathogenesis in humans. Therefore, we investigated the use of piglets as a model for pneumococcal colonization and invasive disease. RESULTS: Intravenous inoculation of piglets with an invasive pneumococcal isolate led to bacteraemia during 5 days, showing clear bacterial replication in the first two days. Bacteraemia was frequently associated with fever and septic arthritis. Moreover, intranasal inoculation of piglets with a nasopharyngeal isolate led to colonization for at least six consecutive days. CONCLUSIONS: This demonstrates that central aspects of human pneumococcal infections can be modelled in piglets enabling the use of this model for studies on colonization and transmission but also on development of vaccines and host-directed therapies. Moreover this is the first example of an animal model inducing high levels of pneumococcal septic arthritis.
Subject(s)
Bacteremia/pathology , Disease Models, Animal , Pneumococcal Infections/veterinary , Streptococcus pneumoniae/pathogenicity , Swine Diseases/microbiology , Animals , Arthritis, Infectious/microbiology , Bacteremia/microbiology , Fever/etiology , Humans , Nasopharynx/microbiology , Pneumococcal Infections/microbiology , Pneumococcal Infections/pathology , Swine , Swine Diseases/pathologyABSTRACT
Using a novel porcine model of intravenous Streptococcus pneumoniae infection, we showed that invasive pneumococcal infections induce marked platelet activation and hyperreactivity. This may contribute to the vascular complications seen in pneumococcal infection.
Subject(s)
Blood Platelets/microbiology , Platelet Activation , Pneumococcal Infections/blood , Pneumococcal Infections/complications , Streptococcus pneumoniae/physiology , Animals , Blood Platelets/pathology , Disease Models, Animal , Humans , Pneumococcal Infections/pathology , Pneumonia, Pneumococcal/blood , Pneumonia, Pneumococcal/complications , Pneumonia, Pneumococcal/pathology , Streptococcus pneumoniae/isolation & purification , SwineABSTRACT
Chaperone and protease systems play essential roles in cellular homeostasis and have vital functions in controlling the abundance of specific cellular proteins involved in processes such as transcription, replication, metabolism and virulence. Bacteria have evolved accurate regulatory systems to control the expression and function of chaperones and potentially destructive proteases. Here, we have used a combination of transcriptomics, proteomics and targeted mutagenesis to reveal that the clp gene regulator (ClgR) of Mycobacterium tuberculosis activates the transcription of at least ten genes, including four that encode protease systems (ClpP1/C, ClpP2/C, PtrB and HtrA-like protease Rv1043c) and three that encode chaperones (Acr2, ClpB and the chaperonin Rv3269). Thus, M. tuberculosis ClgR controls a larger network of protein homeostatic and regulatory systems than ClgR in any other bacterium studied to date. We demonstrate that ClgR-regulated transcriptional activation of these systems is essential for M. tuberculosis to replicate in macrophages. Furthermore, we observe that this defect is manifest early in infection, as M. tuberculosis lacking ClgR is deficient in the ability to control phagosome pH 1 h post-phagocytosis.
Subject(s)
Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Molecular Chaperones/genetics , Mycobacterium tuberculosis/genetics , Peptide Hydrolases/genetics , Regulon , Animals , Bacterial Proteins/genetics , Binding Sites , Cells, Cultured , Gene Deletion , Gene Expression Profiling , Genes, Regulator , Genetic Complementation Test , Macrophages/microbiology , Mice , Mycobacterium tuberculosis/metabolism , Phagosomes/microbiology , Proteomics , Transcriptional ActivationABSTRACT
Inactivation of the transcriptional regulator PhoP results in Mycobacterium tuberculosis attenuation. Preclinical testing has shown that attenuated M. tuberculosis phoP mutants hold promise as safe and effective live vaccine candidates. We focused this study to decipher the virulence networks regulated by PhoP. A combined transcriptomic and proteomic analysis revealed that PhoP controls a variety of functions including: hypoxia response through DosR crosstalking, respiratory metabolism, secretion of the major T-cell antigen ESAT-6, stress response, synthesis of pathogenic lipids and the M. tuberculosis persistence through transcriptional regulation of the enzyme isocitrate lyase. We also demonstrate that the M. tuberculosis phoP mutant SO2 exhibits an antigenic capacity similar to that of the BCG vaccine. Finally, we provide evidence that the SO2 mutant persists better in mouse organs than BCG. Altogether, these findings indicate that PhoP orchestrates a variety of functions implicated in M. tuberculosis virulence and persistence, making phoP mutants promising vaccine candidates.