RESUMEN
Human immunodeficiency virus (HIV) 1 major histocompatibility complex (MHC) I-restricted epitopes are widely believed to be derived from viral proteins encoded by primary open reading frames. However, the HIV-1 genome contains alternative reading frames (ARFs) potentially encoding small polypeptides. We have identified a panel of epitopes encoded by ARFs within the gag, pol, and env genes. The corresponding epitopic peptides were immunogenic in mice humanized for MHC-I molecules. In addition, cytotoxic T lymphocytes recognizing these epitopes were found in HIV-infected patients. These results reveal the existence of atypical mechanisms of HIV-1 epitope generation. They indicate that the repertoire of epitopes recognized by the cellular anti-HIV-1 immune response is broader than initially thought. This should be taken into account when designing vaccine strategies aimed at activating these responses.
Asunto(s)
Antígenos VIH/genética , VIH-1/genética , VIH-1/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Adulto , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , ADN Viral/genética , Epítopos/genética , Femenino , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Datos de Secuencia Molecular , Sistemas de Lectura , Linfocitos T Citotóxicos/inmunologíaRESUMEN
A significant increase in sensitivity to several antibiotics was observed in vitro after infection of the two Pseudomonas aeruginosa strains O1 and K with the filamentous phage Pf3 and Pf1, respectively. Moreover, upon infection with phage Pf1 a P. aeruginosa K strain harboring a plasmid-borne gentamicin resistance gene could be resensitized to the antibiotic. We further show that BALB/c mice were rescued from lethal infections with P. aeruginosa K by concomitant treatment with phage Pf1 and low concentrations of gentamicin, neither of which was able to cure the infection when administered alone.
Asunto(s)
Antibacterianos/uso terapéutico , Gentamicinas/uso terapéutico , Inovirus/fisiología , Infecciones por Pseudomonas/terapia , Fagos Pseudomonas/fisiología , Pseudomonas aeruginosa/virología , Animales , Antibacterianos/farmacología , Bacteriófago Pf1/fisiología , Gentamicinas/farmacología , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidadRESUMEN
Chikungunya virus (CHIKV), a mosquito-transmitted alphavirus, recently reemerged in the Indian Ocean, India and Southeast Asia, causing millions of cases of severe polyarthralgia. No specific treatment to prevent disease or vaccine to limit epidemics is currently available. Here we describe a recombinant live-attenuated measles vaccine (MV) expressing CHIKV virus-like particles comprising capsid and envelope structural proteins from the recent CHIKV strain La Reunion. Immunization of mice susceptible to measles virus induced high titers of CHIKV antibodies that neutralized several primary isolates. Specific cellular immune responses were also elicited. A single immunization with this vaccine candidate protected all mice from a lethal CHIKV challenge, and passive transfer of immune sera conferred protection to naïve mice. Measles vaccine is one of the safest and most effective human vaccines. A recombinant MV-CHIKV virus could make a safe and effective vaccine against chikungunya that deserves to be further tested in human trials.
Asunto(s)
Infecciones por Alphavirus/prevención & control , Virus Chikungunya/inmunología , Vacuna Antisarampión/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Proteínas de la Cápside/inmunología , Fiebre Chikungunya , Chlorocebus aethiops , Reacciones Cruzadas , Sueros Inmunes/inmunología , Inmunidad Celular , Inmunización Pasiva , Ratones , Ratones Transgénicos , Vacunas Atenuadas/inmunología , Células Vero , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Pneumococcus is one of the most important human pathogens that causes life-threatening invasive diseases, especially at the extremities of age. Capsular polysaccharides (CPSs) are known to induce protective antibodies; however, it is not feasible to develop CPS-based vaccines that cover all of the 90 disease-causing serotypes. We applied a genomic approach and described the antibody repertoire for pneumococcal proteins using display libraries expressing 15-150 amino acid fragments of the pathogen's proteome. Serum antibodies of exposed, but not infected, individuals and convalescing patients identified the ANTIGENome of pneumococcus consisting of approximately 140 antigens, many of them surface exposed. Based on several in vitro assays, 18 novel candidates were preselected for animal studies, and 4 of them showed significant protection against lethal sepsis. Two lead vaccine candidates, protein required for cell wall separation of group B streptococcus (PcsB) and serine/threonine protein kinase (StkP), were found to be exceptionally conserved among clinical isolates (>99.5% identity) and cross-protective against four different serotypes in lethal sepsis and pneumonia models, and have important nonredundant functions in bacterial multiplication based on gene deletion studies. We describe for the first time opsonophagocytic killing activity for pneumococcal protein antigens. A vaccine containing PcsB and StkP is intended for the prevention of infections caused by all serotypes of pneumococcus in the elderly and in children.
Asunto(s)
Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/química , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/inmunología , Adulto , Aminoácidos/química , Animales , Anticuerpos , Antígenos Bacterianos/química , Niño , Epítopos/química , Humanos , Inmunoglobulina A/química , Inmunoglobulina G/química , Ratones , Persona de Mediana Edad , Polisacáridos/químicaRESUMEN
Bacteriophage therapy of bacterial infections has received renewed attention owing to the increasing prevalence of antibiotic-resistant pathogens. A side effect of many antibiotics as well as of phage therapy with lytic phage is the release of cell wall components, e.g., endotoxins of gram-negative bacteria, which mediate the general pathological aspects of septicemia. Here we explored an alternative strategy by using genetically engineered nonreplicating, nonlytic phage to combat an experimental Pseudomonas aeruginosa infection. An export protein gene of the P. aeruginosa filamentous phage Pf3 was replaced with a restriction endonuclease gene. This rendered the Pf3 variant (Pf3R) nonreplicative and concomitantly prevented the release of the therapeutic agent from the target cell. The Pf3R phage efficiently killed a wild-type host in vitro, while endotoxin release was kept to a minimum. Treatment of P. aeruginosa infections of mice with Pf3R or with a replicating lytic phage resulted in comparable survival rates upon challenge with a minimal lethal dose of 3. However, the survival rate after phage therapy with Pf3R was significantly higher than that with the lytic phage upon challenge with a minimal lethal dose of 5. This higher survival rate correlated with a reduced inflammatory response elicited by Pf3R treatment relative to that with the lytic phage. Therefore, this study suggests that the increased survival rate of Pf3R-treated mice could result from reduced endotoxin release. Thus, the use of a nonreplicating modified phage for the delivery of genes encoding proteins toxic to bacterial pathogens may open up a new avenue in antimicrobial therapy.
Asunto(s)
Bacteriófago Pf1/genética , Infecciones por Pseudomonas/terapia , Pseudomonas aeruginosa/virología , Animales , Endotoxinas/biosíntesis , Endotoxinas/genética , Interleucina-6/biosíntesis , Ratones , Ratones Endogámicos BALB C , Organismos Modificados Genéticamente , Plásmidos/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Replicación ViralRESUMEN
The Sm-like protein Hfq has been implicated in the regulation of sigmaS-dependent and sigmaS-independent genes in E. coli and in the regulation of virulence factors in both, Yersinia enterocolitica and Brucella abortus. Here, we have studied the effect of Hfq on virulence and stress response of Pseudomonas aeruginosa (PAO1). We have constructed a PAO1hfq- mutant and a PAO1hfq-rpoS- double mutant to permit distinction between direct and indirect effects of Hfq. When compared to the wild-type and the rpoS- strains, the hfq knock out strain showed a reduced growth rate and was unable to utilize glucose as a sole carbon source. Elastase activity was 80% reduced in the hfq- mutant when compared to the wild-type or the rpoS- strain, whereas alginate production seemed to be solely affected by sigmaS. The production of catalase and pyocyanin was shown to be affected in an additive manner by both, Hfq and sigmaS. Moreover, twitching and swarming mediated by typeIV pili was shown to be impaired in the hfq- mutant. When compared to PAO1 wild-type and the rpoS- mutant, the hfq- mutant decreased virulence in Galleria mellonella by a factor of 1 x 10(4) and 5 x 10(3), respectively. Likewise, when compared to wild-type, the PAO1hfq- mutant was significantly attenuated in virulence when administered intraperitoneally in mice. These results strongly suggest that Hfq is a global regulator of PAO1 virulence and stress response which is not exclusively due to its role in stimulating the synthesis of sigmaS.