RESUMO
A critical global health need exists for a Zika vaccine capable of mitigating the effects of future Zika epidemics. In this study we evaluated the antibody responses and efficacy of an aluminum hydroxide adjuvanted purified inactivated Zika vaccine (PIZV) against challenge with Zika virus (ZIKV) strain PRVABC59. Indian rhesus macaques received two doses of PIZV at varying concentrations ranging from 0.016 µg - 10 µg and were subsequently challenged with ZIKV six weeks or one year following the second immunization. PIZV induced a dose-dependent immune response that was boosted by a second immunization. Complete protection against ZIKV infection was achieved with the higher PIZV doses of 0.4 µg, 2 µg, and 10 µg at 6 weeks and with 10 ug PIZV at 1 year following vaccination. Partial protection was achieved with the lower PIZV doses of 0.016 µg and 0.08 µg. Based on these data, a neutralizing antibody response above 3.02 log10 EC50 was determined as a correlate of protection in macaques. PIZV elicited a dose-dependent neutralizing antibody response which is protective for at least 1 year following vaccination.
Assuntos
Vacinas de Produtos Inativados , Vacinas Virais , Infecção por Zika virus , Zika virus , Animais , Feminino , Masculino , Anticorpos Neutralizantes/sangue , Modelos Animais de Doenças , Imunoglobulina G/sangue , Macaca , RNA Viral/imunologia , RNA Viral/metabolismo , Vacinação , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/imunologia , Zika virus/imunologia , Zika virus/patogenicidade , Infecção por Zika virus/patologia , Infecção por Zika virus/prevenção & controleRESUMO
Fusobacterium necrophorum, a Gram-negative anaerobe, causes a variety of necrotic infections in humans and animals. There are two subspecies: subsp. necrophorum and subsp. funduliforme. In cattle, subsp. necrophorum is more prevalent and production of leukotoxin is a major virulence factor. The leukotoxin operon (lktBAC) consists of three genes, lktB, lktA and lktC, of which lktA is the structural toxin gene. The subspecies identity of human F. necrophorum is less certain and it is not known whether human strains possess the leukotoxin gene or leukotoxin activity. Therefore, the objective of this study was to identify the subspecies status of four human clinical strains of F. necrophorum and determine whether they have the leukotoxin gene or leukotoxin activity. Phenotypic and genotypic characteristics suggested that the four strains belonged to subsp. funduliforme, which was confirmed by sequencing the 16S rRNA gene. Analysis of the four strains by PCR revealed the presence of the leukotoxin operon. Partial DNA sequencing identified one human strain with full-length lktA, whereas the others exhibited considerable heterogeneity in size. All strains had a leukotoxin operon promoter-containing intergenic region similar to that of bovine subsp. funduliforme strains, which was confirmed by DNA sequencing and Southern blotting. Despite variations in the lktA gene, all strains secreted leukotoxin as demonstrated by Western blotting. Flow cytometry assays revealed that the leukotoxin was toxic to human white blood cells. In conclusion, the human strains examined contained a leukotoxin gene whose gene product was biologically active. The importance of leukotoxin as a virulence factor in human fusobacterial infections needs further evaluation.
Assuntos
Exotoxinas/genética , Exotoxinas/toxicidade , Fusobacterium necrophorum/genética , Fusobacterium necrophorum/patogenicidade , Leucócitos/microbiologia , Fatores de Virulência/biossíntese , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Southern Blotting , Western Blotting , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Intergênico , DNA Ribossômico/química , DNA Ribossômico/genética , Exotoxinas/biossíntese , Citometria de Fluxo , Infecções por Fusobacterium/microbiologia , Fusobacterium necrophorum/isolamento & purificação , Proteínas Hemolisinas/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Genético , Regiões Promotoras Genéticas , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Liver abscesses in cattle are associated primarily with Fusobacterium necrophorum, a gram negative, pleomorphic, and obligate anaerobe. In cattle, the organism is a normal inhabitant of the rumen and an opportunistic pathogen. There are two subspecies: subsp. necrophorum and subsp. funduliforme. Subspecies necrophorum is more frequently isolated, often in pure culture, from liver abscesses than subspecies funduliforme. Leukotoxin (Lkt), an exotoxin, is a major virulence factor. In subsp. necrophorum, Lkt is a high molecular weight protein that is encoded by a tricistronic leukotoxin operon (lktBAC) and induces apoptosis and necrosis of bovine leukocytes in a dose-dependent manner. The subsp. funduliforme produces lower concentration of leukotoxin and hence less virulent than subsp. necrophorum. The probable cause of low leukotoxin production by subsp. funduliforme is not known. We sequenced the leukotoxin operon and compared it to the operon of subsp. necrophorum. The lkt operon had three genes, lktB, lktA, and lktC and was similar in organization to that of subsp. necrophorum. The subsp. funduliforme LktB and LktA proteins had significant differences in their N-terminal sequences despite high overall amino acid similarities, 87% and 88%, respectively with subsp. necrophorum. The relative expressions of lktA in both subspecies at various growth phases were determined by quantitative PCR (Q-PCR). Data from Q-PCR studies revealed that subsp. funduliforme had a 21.1-fold lower lktA transcript level in mid-log phase cells than subsp. necrophorum. The lktA transcript amounts were lower in all stages of growth in subsp. funduliforme. The maximum concentration of leukotoxin and the highest cytotoxicity on bovine PMNs were observed in the mid-log phase, which corresponded to the highest amount of lktA transcript detected. Therefore, the low toxicity associated with subsp. funduliforme leukotoxin, a less virulent subspecies, may in part be due to the differences in the lktA gene and reduced transcription.
Assuntos
Exotoxinas/genética , Exotoxinas/toxicidade , Fusobacterium necrophorum/genética , Óperon , Animais , Bovinos , Sobrevivência Celular , Exotoxinas/imunologia , Exotoxinas/metabolismo , Fusobacterium necrophorum/crescimento & desenvolvimento , Fusobacterium necrophorum/imunologia , Fusobacterium necrophorum/patogenicidade , Expressão Gênica , Leucócitos Mononucleares/citologiaRESUMO
Fusobacterium equinum, a gram negative, rod-shaped and an obligate anaerobic bacterium is a newly described species. The organism is associated with necrotic infections of the respiratory tract in horses that include necrotizing pneumonia, pleuritis and paraoral infections. The species is closely related to F. necrophorum that causes liver abscesses in cattle and sheep, calf-diphtheria in cattle, and foot-rot in sheep and cattle. Leukotoxin, an exotoxin, is an important virulence factor in bovine strains of F. necrophorum. Our objective was to examine strains (n=10) of F. equinum for leukotoxin (lktA) gene and its toxic effects on equine leukocytes. Southern hybridization and partial DNA sequencing revealed that all the 10 strains had the lktA gene with greater similarities to F. necrophorum subsp. necrophorum. The secreted leukotoxin was detected in the culture supernatant and its biological activity was determined by viability assays with equine polymorphonuclear cells (PMNs) using flow cytometry. While culture supernatants of four strains (E1, E7, E9, and E10) were highly toxic to equine PMNs; strain E5 was moderately toxic and the remaining strains (E2, E3, E4, E6, and E8) were only mildly toxic. Our data indicated that F. equinum isolates had lktA gene and its product was toxic to equine leukocytes. Therefore, leukotoxin may be an important virulence factor in F. equinum infections.
