RESUMEN
We used a replication-incompetent, single-cycle, alphavirus replicon vector system to produce virus-like replicon particles (VRP) expressing the extracellular domain of human cytomegalovirus (CMV) glycoprotein B or a pp65/IE1 fusion protein. Efficient production methods were scaled to produce pilot lots and clinical lots of each alphavirus replicon vaccine component. The vaccine induced high-titered antibody responses in mice and rabbits, as measured by ELISA and CMV neutralization assays, and robust T-cell responses in mice, as measured by IFN-gamma ELISPOT assay. A toxicity study in rabbits showed no adverse effects in any toxicology parameter. These studies support clinical testing of this novel CMV alphavirus replicon vaccine in humans.
Asunto(s)
Alphavirus/genética , Vacunas contra Citomegalovirus/genética , Citomegalovirus/genética , Citomegalovirus/inmunología , Animales , Anticuerpos Antivirales/sangre , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/prevención & control , Vacunas contra Citomegalovirus/inmunología , Vacunas contra Citomegalovirus/toxicidad , Femenino , Vectores Genéticos , Humanos , Inmunidad Celular , Masculino , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Conejos , Replicón , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/toxicidadRESUMEN
Development of vaccines against cytomegalovirus (CMV) is an important public health priority. We used a propagation-defective, single-cycle RNA replicon vector system derived from an attenuated strain of an alphavirus, Venezuelan equine encephalitis virus, to produce virus-like replicon particles (VRP) expressing various combinations of pp65, IE1, or gB proteins of human CMV. Protein expression in VRP-infected cells was highest with single-promoter replicons expressing pp65, IE1, a pp65/IE1 fusion protein, or the extracellular domain of gB and with double-promoter replicons expressing pp65 and IE1. Protein expression was lower with double- and triple-promoter replicons expressing gB, especially the full-length form of gB. BALB/c mice immunized with VRP expressing gB developed high titers of neutralizing antibody to CMV, and mice immunized with VRP expressing pp65, IE1, or a pp65/IE1 fusion protein developed robust antigen-specific T-cell responses as measured by gamma interferon enzyme-linked immunospot assay. Three overlapping immunodominant pp65 peptides contained a nine-amino-acid sequence (LGPISGHVL) that matches the consensus binding motif for a major histocompatibility complex H2-D(d) T-cell epitope. These data provide the basis for further development and clinical evaluation of an alphavirus replicon vaccine for CMV expressing the pp65, IE1, and gB proteins.
Asunto(s)
Formación de Anticuerpos , Citomegalovirus/inmunología , Proteínas Inmediatas-Precoces/inmunología , Inmunidad Celular , Fosfoproteínas/inmunología , Replicón/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas de la Matriz Viral/inmunología , Proteínas Virales/inmunología , Vacunas Virales/inmunología , Animales , Chlorocebus aethiops , Femenino , Humanos , Inmunización/métodos , Inmunización Secundaria , Ratones , Ratones Endogámicos BALB C , Células VeroRESUMEN
Recombinant severe acute respiratory syndrome (SARS) nucleocapsid and spike protein-based immunoglobulin G immunoassays were developed and evaluated. Our assays demonstrated high sensitivity and specificity to the SARS coronavirus in sera collected from patients as late as 2 years postonset of symptoms. These assays will be useful not only for routine SARS coronavirus diagnostics but also for epidemiological and antibody kinetic studies.
Asunto(s)
Anticuerpos Antivirales/sangre , Glicoproteínas de Membrana/inmunología , Proteínas de la Nucleocápside/inmunología , Síndrome Respiratorio Agudo Grave/diagnóstico , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Proteínas del Envoltorio Viral/inmunología , Proteínas de la Nucleocápside de Coronavirus , Ensayo de Inmunoadsorción Enzimática , Humanos , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Pruebas Serológicas , Glicoproteína de la Espiga del CoronavirusRESUMEN
HIV-1 genetic diversity among circulating strains presents a major challenge for HIV-1 vaccine development, particularly for developing countries where less sequence information is available. To identify representative viruses for inclusion in candidate vaccines targeted for South Africa, we applied an efficient sequence survey strategy to samples from recently and chronically infected persons residing in potential vaccine trial sites. All 111 sequences were subtype C, including 30 partial gag, 26 partial pol, 27 V2-V3 env, and 28 V5-partial gp41 sequences. Of the 10 viruses cultured from recently infected individuals, 9 were R5 and 1 was R5X4. Two isolates, Du151 and Du422, collected within 2 months of infection, were selected as vaccine strains on the basis of their amino acid similarity to a derived South African consensus sequence The selection of recently transmitted R5 isolates for vaccine design may provide an advantage in a subtype C R5-dominant epidemic. The full-length Du422 gag and Du151 pol and env genes were cloned into the Venezuelan equine encephalitis (VEE) replicon particle (VRP) expression system. Du422 Gag protein expressed from the VRP accumulated to a high level and was immunogenic as demonstrated by cytotoxic T lymphocyte responses in mice vaccinated with gag-VRPs. Optimization of codon use for VRP expression in human cells did not enhance expression of the gag gene. The cloned Du151 env gene encoded a functional protein as demonstrated by fusion of VRP-infected cells with cells expressing CD4 and CCR5. Genes identified in this study have been incorporated into the VEE VRP candidate vaccines targeted for clinical trial in South Africa.
Asunto(s)
Vacunas contra el SIDA , Infecciones por VIH/prevención & control , VIH-1/clasificación , VIH-1/aislamiento & purificación , Animales , Línea Celular , Chlorocebus aethiops , Ensayos Clínicos como Asunto , Virus de la Encefalitis Equina Venezolana/genética , Productos del Gen env/genética , Productos del Gen env/metabolismo , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Vectores Genéticos , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Filogenia , Replicón/genética , Análisis de Secuencia de ADN , Sudáfrica , Células Vero , Virión/genética , Virión/metabolismoRESUMEN
Alphaviruses are positive-stranded RNA viruses that have a broad host range and therefore are capable of replicating in many vertebrate and invertebrate cells. The single-stranded alphavirus genome is divided into two ORFs. The first ORF encodes the nonstructural proteins that are translated upon entry of the virus into the cytoplasm and are responsible for transcription and replication of viral RNA. The second ORF is under the control of a subgenomic promoter and normally encodes the structural proteins, which are responsible for encapsidation of viral RNA and final assembly into enveloped particles. Expression vectors have been engineered from at least three alphaviruses in which the structural protein gene region has been replaced by heterologous genes and have been shown to express high levels of the heterologous protein in cultured cells. These RNA vectors, known as replicons, are capable of replicating on their own but are not packaged into virus-like particles unless the structural proteins are provided in trans. Thus, replicons are single cycle vectors incapable of spreading from infected to noninfected cells. Because of these features, alphavirus replicon vectors are being developed as a platform vaccine technology for numerous viral, bacterial, protozoan and tumour antigens where they have been shown to be efficient inducers of both humoral and T cell responses. In addition, as the alphavirus structural proteins are not expressed in vaccine recipients, antivector immune responses are generally minimal, allowing for multiple effective immunisations of the same individual.