RESUMEN
UNLABELLED: Human cytomegalovirus (HCMV) is the major viral cause of birth defects and a serious problem in immunocompromised individuals and has been associated with atherosclerosis. Previous studies have shown that the induction of autophagy can inhibit the replication of several different types of DNA and RNA viruses. The goal of the work presented here was to determine whether constitutive activation of autophagy would also block replication of HCMV. Most prior studies have used agents that induce autophagy via inhibition of the mTOR pathway. However, since HCMV infection alters the sensitivity of mTOR kinase-containing complexes to inhibitors, we sought an alternative method of inducing autophagy. We chose to use trehalose, a nontoxic naturally occurring disaccharide that is found in plants, insects, microorganisms, and invertebrates but not in mammals and that induces autophagy by an mTOR-independent mechanism. Given the many different cell targets of HCMV, we proceeded to determine whether trehalose would inhibit HCMV infection in human fibroblasts, aortic artery endothelial cells, and neural cells derived from human embryonic stem cells. We found that in all of these cell types, trehalose induces autophagy and inhibits HCMV gene expression and production of cell-free virus. Treatment of HCMV-infected neural cells with trehalose also inhibited production of cell-associated virus and partially blocked the reduction in neurite growth and cytomegaly. These results suggest that activation of autophagy by the natural sugar trehalose or other safe mTOR-independent agents might provide a novel therapeutic approach for treating HCMV disease. IMPORTANCE: HCMV infects multiple cell types in vivo, establishes lifelong persistence in the host, and can cause serious health problems for fetuses and immunocompromised individuals. HCMV, like all other persistent pathogens, has to finely tune its interplay with the host cellular machinery to replicate efficiently and evade detection by the immune system. In this study, we investigated whether modulation of autophagy, a host pathway necessary for the recycling of nutrients and removal of protein aggregates, misfolded proteins, and pathogens, could be used to target HCMV. We found that autophagy could be significantly increased by treatment with the nontoxic, natural disaccharide trehalose. Importantly, trehalose had a profound inhibitory effect on viral gene expression and strongly impaired viral spread. These data constitute a proof-of-concept for the use of natural products targeting host pathways rather than the virus itself, thus reducing the risk of the development of resistance to treatment.
Asunto(s)
Autofagia/efectos de los fármacos , Citomegalovirus/fisiología , Trehalosa/metabolismo , Replicación Viral , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/virología , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Humanos , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/virologíaRESUMEN
25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.
Asunto(s)
Hidroxicolesteroles/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Animales , Células de la Médula Ósea/citología , Ésteres del Colesterol/metabolismo , Perfilación de la Expresión Génica , Hidroxicolesteroles/metabolismo , Receptores X del Hígado , Macrófagos/citología , Macrófagos/virología , Ratones , Ratones Endogámicos C57BL , Muromegalovirus/fisiología , Receptores Nucleares Huérfanos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Esfingolípidos/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidoresRESUMEN
Leukemia cells from patients with chronic lymphocytic leukemia (CLL) express a highly restricted immunoglobulin heavy variable chain (IGHV) repertoire, suggesting that a limited set of antigens reacts with leukemic cells. Here, we evaluated the reactivity of a panel of different CLL recombinant antibodies (rAbs) encoded by the most commonly expressed IGHV genes with a panel of selected viral and bacterial pathogens. Six different CLL rAbs encoded by IGHV1-69 or IGHV3-21, but not a CLL rAb encoded by IGHV4-39 genes, reacted with a single protein of human cytomegalovirus (CMV). The CMV protein was identified as the large structural phosphoprotein pUL32. In contrast, none of the CLL rAbs bound to any other structure of CMV, adenovirus serotype 2, Salmonella enterica serovar Typhimurium, or of cells used for propagation of these microorganisms. Monoclonal antibodies or humanized rAbs of irrelevant specificity to pUL32 did not react with any of the proteins present in the different lysates. Still, rAbs encoded by a germ line IGHV1-69 51p1 allele from CMV-seropositive and -negative adults also reacted with pUL32. The observed reactivity of multiple different CLL rAbs and natural antibodies from CMV-seronegative adults with pUL32 is consistent with the properties of a superantigen.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Citomegalovirus/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Fosfoproteínas/inmunología , Superantígenos/inmunología , Adulto , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos/inmunología , Linfocitos B/metabolismo , Western Blotting , Células Cultivadas , Células HEK293 , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/metabolismo , Unión Proteica/inmunología , Proteínas Recombinantes/inmunología , Salmonella typhimurium/inmunología , Proteínas Virales/inmunologíaRESUMEN
To date, no vaccine that is safe and effective against herpes simplex virus 2 (HSV-2) disease has been licensed. In this study, we evaluated a DNA prime-formalin-inactivated-HSV-2 (FI-HSV2) boost vaccine approach in the guinea pig model of acute and recurrent HSV-2 genital disease. Five groups of guinea pigs were immunized and intravaginally challenged with HSV-2. Two groups were primed with plasmid DNAs encoding the secreted form of glycoprotein D2 (gD2t) together with two genes required for viral replication, either the helicase (UL5) and DNA polymerase (UL30) genes or the single-stranded DNA binding protein (UL29) and primase (UL52) genes. Both DNA-primed groups were boosted with FI-HSV2 formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Two additional groups were primed with the empty backbone plasmid DNA (pVAX). These two groups were boosted with MPL and alum (MPL-alum) together with either formalin-inactivated mock HSV-2 (FI-Mock) or with FI-HSV2. The final group was immunized with gD2t protein in MPL-alum. After challenge, 0/9 animals in the group primed with UL5, UL30, and gD2t DNAs and all 10 animals in the mock-immunized control group (pVAX-FI-Mock) developed primary lesions. All mock controls developed recurrent lesions through day 100 postchallenge. Only 1 guinea pig in the group primed with pVAX DNA and boosted with FI-HSV2 (pVAX-FI-HSV2 group) and 2 guinea pigs in the group primed with UL5, UL30, and gD2t DNAs and boosted with FI-HSV2 (UL5, UL30, gD2t DNA-FI-HSV2 group) developed recurrent lesions. Strikingly, the UL5, UL30, gD2t DNA-FI-HSV2 group showed a 97% reduction in recurrent lesion days compared with the mock controls, had the highest reduction in days with recurrent disease, and contained the lowest mean HSV-2 DNA load in the dorsal root ganglia.
Asunto(s)
Herpes Genital/prevención & control , Herpesvirus Humano 2/inmunología , Vacunas contra Herpesvirus/inmunología , Inmunización Secundaria/métodos , Vacunación/métodos , Vacunas de ADN/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , ADN Viral/genética , Femenino , Ganglios Espinales/virología , Cobayas , Herpes Genital/inmunología , Herpes Genital/patología , Herpesvirus Humano 2/genética , Vacunas contra Herpesvirus/administración & dosificación , Lípido A/administración & dosificación , Lípido A/análogos & derivados , Prevención Secundaria , Vacunas de ADN/administración & dosificación , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunologíaRESUMEN
Zika virus (ZIKV) is a mosquito-borne flavivirus with maternal infection associated with preterm birth, congenital malformations, and fetal death, and adult infection associated with Guillain-Barré syndrome. Recent widespread endemic transmission of ZIKV and the potential for future outbreaks necessitate the development of an effective vaccine. We developed a ZIKV vaccine candidate based on virus-like-particles (VLPs) generated following transfection of mammalian HEK293T cells using a plasmid encoding the pre-membrane/membrane (prM/M) and envelope (E) structural protein genes. VLPs were collected from cell culture supernatant and purified by column chromatography with yields of approximately 1-2mg/L. To promote increased particle yields, a single amino acid change of phenylalanine to alanine was made in the E fusion loop at position 108 (F108A) of the lead VLP vaccine candidate. This mutation resulted in a modest 2-fold increase in F108A VLP production with no detectable prM processing by furin to a mature particle, in contrast to the lead candidate (parent). To evaluate immunogenicity and efficacy, AG129 mice were immunized with a dose titration of either the immature F108A or lead VLP (each alum adjuvanted). The resulting VLP-specific binding antibody (Ab) levels were comparable. However, geometric mean neutralizing Ab (nAb) titers using a recombinant ZIKV reporter were significantly lower with F108A immunization compared to lead. After virus challenge, all lead VLP-immunized groups showed a significant 3- to 4-Log10 reduction in mean ZIKV RNAemia levels compared with control mice immunized only with alum, but the RNAemia reduction of 0.5 Log10 for F108A groups was statistically similar to the control. Successful viral control by the lead VLP candidate following challenge supports further vaccine development for this candidate. Notably, nAb titer levels in the lead, but not F108A, VLP-immunized mice inversely correlated with RNAemia. Further evaluation of sera by an in vitro Ab-dependent enhancement assay demonstrated that the F108A VLP-induced immune sera had a significantly higher capacity to promote ZIKV infection in FcγR-expressing cells. These data indicate that a single amino acid change in the fusion loop resulted in increased VLP yields but that the immature F108A particles were significantly diminished in their capacity to induce nAbs and provide protection against ZIKV challenge.
Asunto(s)
Nacimiento Prematuro , Vacunas de Partículas Similares a Virus , Vacunas Virales , Infección por el Virus Zika , Virus Zika , Aminoácidos , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Femenino , Células HEK293 , Humanos , Recién Nacido , Mamíferos , Ratones , Mutación , Virus Zika/genéticaRESUMEN
BACKGROUND: Zika virus (ZIKV), a mosquito-borne flavivirus, is a re-emerging virus that constitutes a public health threat due to its recent global spread, recurrent outbreaks, and infections that are associated with neurological abnormalities in developing fetuses and Guillain-Barré syndrome in adults. To date, there are no approved vaccines against ZIKV infection. Various preclinical and clinical development programs are currently ongoing in an effort to bring forward a vaccine for ZIKV. METHODOLOGY/PRINCIPLE FINDINGS: We have developed a ZIKV vaccine candidate based on Virus-Like-Particles (VLPs) produced in HEK293 mammalian cells using the prM (a precursor to M protein) and envelope (E) structural protein genes from ZIKV. Transient transfection of cells via plasmid and electroporation produced VLPs which were subsequently purified by column chromatography yielding approximately 2mg/L. Initially, immunogenicity and efficacy were evaluated in AG129 mice using a dose titration of VLP with and without Alhydrogel 2% (alum) adjuvant. We found that VLP with and without alum elicited ZIKV-specific serum neutralizing antibodies (nAbs) and that titers correlated with protection. A follow-up immunogenicity and efficacy study in rhesus macaques was performed using VLP formulated with alum. Multiple neutralization assay methods were performed on immune sera including a plaque reduction neutralization test, a microneutralization assay, and a Zika virus Renilla luciferase neutralization assay. All of these assays indicate that following immunization, VLP induces high titer nAbs which correlate with protection against ZIKV challenge. CONCLUSIONS/SIGNIFICANCE: These studies confirm that ZIKV VLPs could be efficiently generated and purified. Upon VLP immunization, in both mice and NHPs, nAb was induced that correlate with protection against ZIKV challenge. These studies support translational efforts in developing a ZIKV VLP vaccine for evaluation in human clinical trials.
Asunto(s)
Vacunas de Partículas Similares a Virus/inmunología , Vacunas Virales/inmunología , Infección por el Virus Zika/prevención & control , Virus Zika/inmunología , Adyuvantes Inmunológicos/farmacología , Compuestos de Alumbre/farmacología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Femenino , Células HEK293 , Humanos , Macaca mulatta , Masculino , Ratones , Pruebas de Neutralización , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas Virales/administración & dosificación , Infección por el Virus Zika/inmunologíaRESUMEN
Infection with Herpes Simplex Viruses (HSVs) represents a significant health burden worldwide with HSV-1 and HSV-2 causing genital disease and HSV-2 contributing to human immunodeficiency virus acquisition. Despite great need, there is currently no licensed vaccine against HSV. In this report, we evaluated the protective efficacy of a vaccine containing highly purified, inactivated HSV-2 particles (with and without additional recombinant glycoprotein D) formulated with a monophosphoryl lipid A/Alhydrogel adjuvant in a guinea pig HSV genital model. The key results from 3 independent studies were: (1) vaccination consistently provided significant 3-3.5 Log10 reductions in vaginal HSV-2 titers on day 2 postchallenge; (2) following homologous or heterologous challenge with two U.S. isolates, all vaccine groups showed complete protection against lesion formation, significant 3 Log10 reductions in day 2 virus shedding, enhanced virus clearance, significant reductions in HSV-2 DNA within ganglia, and no detectable shedding (<2 PFU) or latent viral DNA in some immunized animals; (3) following challenge with a third heterologous strain, vaccination provided complete protection against primary and recurrent lesions, significant reductions in primary virus shedding, a 50% reduction in recurrent shedding days, and undetectable latent virus in the ganglia and spinal cords of most animals; and (4) adding glycoprotein D provided no enhanced protection relative to that elicited by the inactivated HSV-2 particles alone. Together, these data provide strong support for further development of this exceedingly protective and highly feasible vaccine candidate for human trials.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Herpes Genital/prevención & control , Vacunas contra el Virus del Herpes Simple/administración & dosificación , Herpesvirus Humano 2/efectos de los fármacos , Virión , Administración Intravaginal , Animales , Chlorocebus aethiops , Femenino , Cobayas , Herpes Genital/inmunología , Vacunas contra el Virus del Herpes Simple/inmunología , Herpesvirus Humano 2/inmunología , Inmunidad Humoral/efectos de los fármacos , Inmunidad Humoral/inmunología , Células Vero , Virión/inmunología , Esparcimiento de Virus/efectos de los fármacos , Esparcimiento de Virus/inmunologíaRESUMEN
The human cytomegalovirus (HCMV) IE2 86 protein is essential for viral replication. Two other proteins, IE2 60 and IE2 40, which arise from the C-terminal half of IE2 86, are important for later stages of the infection. Functional analyses of IE2 86 in the context of the infection have utilized bacterial artificial chromosomes as vectors to generate mutant viruses. One limitation is that many mutations result in debilitated or nonviable viruses. Here, we describe a novel system that allows tightly controlled temporal expression of the IE2 proteins and provides complementation of both growth-impaired and nonviable IE2 mutant viruses. The strategy involves creation of cell lines with separate lentiviruses expressing a bicistronic RNA with a selectable marker as the first open reading frame (ORF) and IE2 86, IE2 60, or IE2 40 as the second ORF. Induction of expression of the IE2 proteins occurs only following DNA recombination events mediated by Cre and FLP recombinases that delete the first ORF. HCMV encodes Cre and FLP, which are expressed at immediate-early (for IE2 86) and early-late (for IE2 40 and IE2 60) times, respectively. We show that the presence of full-length IE2 86 alone provides some complementation for virus production, but the correct temporal expression of IE2 86 and IE2 40 together has the most beneficial effect for early-late gene expression and synthesis of infectious virus. This approach for inducible protein translation can be used for complementation of other mutations as well as controlled expression of toxic cellular and microbial proteins.
Asunto(s)
Línea Celular , Citomegalovirus/crecimiento & desarrollo , Prueba de Complementación Genética/métodos , Proteínas Inmediatas-Precoces/genética , Transactivadores/genética , Virología/métodos , Replicación Viral/genética , Citomegalovirus/genética , Eliminación de Gen , Expresión Génica , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Biosíntesis de Proteínas , Recombinación Genética , Factores de Tiempo , Transactivadores/metabolismoRESUMEN
Herpes Simplex Virus Type 2 (HSV-2) infection can result in life-long recurrent genital disease, asymptomatic virus shedding, and transmission. No vaccine to date has shown significant protection clinically. Here, we used a mouse model of genital HSV-2 infection to test the efficacy of a vaccine consisting of whole, formalin-inactivated HSV-2 (FI-HSV2) formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Vaccine components were administered alone or as a prime-boost immunization together with DNA vaccines encoding a truncated glycoprotein D2 (gD2t) and two conserved HSV-2 genes necessary for virus replication, UL5 (DNA helicase) and UL30 (DNA polymerase). Our results show: (1) compared with mock immunized controls, mice immunized with FI-HSV2 plus MPL/alum consistently showed protection against disease burden and total viral shedding while the mice immunized with gD2t protein with MPL/alum did not; (2) protection against genital disease and viral replication correlated with the type of boost in a prime-boost immunization with little advantage afforded by a DNA prime; (3) intramuscular (i.m.) immunization with FI-HSV2 in MPL/Alhydrogel adjuvant provided nearly complete protection against vaginal HSV-2 shedding after a lethal intravaginal (i.vag.) short-term challenge and long-term rechallenge; (4) single formulation immunization with DNA vaccines, FI-HSV2, and MPL in an aluminum phosphate (Adju-Phos) adjuvant did not increase protection relative to FI-HSV2/MPL/Adju-Phos alone; and (5) addition of MPL/alum to the FI-HSV2 was required for optimal protection against disease, viral replication, and latent virus load in the dorsal root ganglia (DRG). Most notably, an optimized vaccine formulation of FI-HSV2 MPL/Alhydrogel given i.m. completely protected against detectable vaginal HSV-2 shedding in the majority of animals and HSV-2 latent DNA in the DRG of all animals.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Herpes Genital/prevención & control , Herpesvirus Humano 2/inmunología , Vacunas contra Herpesvirus/inmunología , Lípido A/análogos & derivados , Esparcimiento de Virus , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Modelos Animales de Enfermedad , Femenino , Ganglios Espinales/virología , Herpes Genital/inmunología , Herpes Genital/patología , Herpes Genital/virología , Herpesvirus Humano 2/genética , Vacunas contra Herpesvirus/administración & dosificación , Lípido A/administración & dosificación , Ratones , Ratones Endogámicos BALB C , Análisis de Supervivencia , Vacunación/métodos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vagina/virologíaRESUMEN
Human cytomegalovirus (HCMV) establishes a lifelong infection with the potential for reinfection or viral transmission even in the presence of strong and diverse CD8 T-lymphocyte responses. This suggests that the CMVs skew the host T-cell response in order to favor viral persistence. In this study, we hypothesized that the essential, nonstructural proteins that are highly conserved among the CMVs may represent a novel class of T-cell targets for vaccine-mediated protection due to their requirements for expression and sequence stability, but that the observed subdominance of these antigens in the CMV-infected host results from the virus limiting the T-cell responses to otherwise-protective specificities. We found that DNA immunization of mice with the murine CMV (MCMV) homologs of HCMV DNA polymerase (M54) or helicase (M105) was protective against virus replication in the spleen following systemic challenge, with the protection level elicited by the M54 DNA being comparable to that of DNA expressing the immunodominant IE1 (pp89). Intracellular gamma interferon staining of CD8 T cells from mice immunized with either the M54 or M105 DNAs showed strong primary responses that recalled rapidly after viral challenge. M54- and M105-specific CD8 T cells were detected after the primary MCMV infection, but their levels were not consistently above the background level. The conserved, essential proteins of the CMVs thus represent a novel class of CD8 T-cell targets that may contribute to a successful HCMV vaccine strategy.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Citomegalovirus/genética , ADN Polimerasa Dirigida por ADN/genética , ADN/administración & dosificación , Proteínas Virales/genética , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular , Citomegalovirus/inmunología , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Human CMV establishes a lifelong latent infection in the majority of people worldwide. Although most infections are asymptomatic, immunocompetent hosts devote an extraordinary amount of immune resources to virus control. To increase our understanding of CMV immunobiology in an animal model, we used a genomic approach to comprehensively map the C57BL/6 CD8 T cell response to murine CMV (MCMV). Responses to 27 viral proteins were detectable directly ex vivo, the most diverse CD8 T cell response yet described within an individual animal. Twenty-four peptide epitopes were mapped from 18 Ags, which together account for most of the MCMV-specific response. Most Ags were from genes expressed at early times, after viral genes that interfere with Ag presentation are expressed, consistent with the hypothesis that the CD8 T cell response to MCMV is largely driven by cross-presented Ag. Titration of peptide epitopes in a direct ex vivo intracellular cytokine staining assay revealed a wide range of functional avidities, with no obvious correlation between functional avidity and the strength of the response. The immunodominance hierarchy varied only slightly between mice and between experiments. However, H-2(b)-expressing mice with different genetic backgrounds responded preferentially to different epitopes, indicating that non-MHC-encoded factors contribute to immunodominance in the CD8 T cell response to MCMV.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD8-positivos/inmunología , Genoma Viral/genética , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Muromegalovirus/genética , Muromegalovirus/inmunología , Enfermedad Aguda , Algoritmos , Secuencia de Aminoácidos , Animales , Antígenos Virales/genética , Computadores , Epítopos/química , Epítopos/inmunología , Biblioteca de Genes , Antígenos de Histocompatibilidad/inmunología , Ratones , Datos de Secuencia Molecular , Muromegalovirus/fisiología , Sistemas de Lectura Abierta/genética , Péptidos/química , Péptidos/inmunologíaRESUMEN
We previously demonstrated that vaccination of BALB/c mice with a pool of 13 plasmid DNAs (pDNAs) expressing murine cytomegalovirus (MCMV) genes followed by formalin-inactivated MCMV (FI-MCMV) resulted in complete protection against viral replication in the spleen and salivary glands following sublethal intraperitoneal (i.p.) challenge. Here, we found that following intranasal (i.n.) challenge, titers of virus in the lungs of the immunized mice were reduced approximately 1,000-fold relative to those for mock-immunized controls. We next sought to extend these results and to determine whether similar protection levels could be achieved by priming with a pool of three pDNAs containing three key plasmids (IE1, M84, and gB). We found that the three-pDNA priming elicited IE1- and M84-p65-specific CD8+ T lymphocytes and, following FI-MCMV boost, high levels of virion-specific immunoglobulin G (IgG) and virus-neutralizing antibodies. When mice were i.n. challenged 4 months after the last boost, titers of virus in the lungs of immunized mice were reduced 1,000- to 2,000-fold from those for controls during the peak of viral replication. Additionally, titers of virus were either at or below the detection limits for the salivary glands, liver, and spleen of the majority of the immunized mice. Following sublethal i.p. challenge, virus was undetectable in all of the above target organs of the immunized mice. Virion-specific IgA in the lungs was consistently detected by day 6 post-i.n. challenge for the immunized mice and by day 14 for controls. These results demonstrate the immunity and high levels of protection of the priming-boosting vaccination against both systemic and mucosal challenge.
Asunto(s)
Vacunas contra Citomegalovirus , Infecciones por Herpesviridae/prevención & control , Muromegalovirus/inmunología , Plásmidos , Vacunas de ADN , Administración Intranasal , Animales , Linfocitos T CD8-positivos/inmunología , Vacunas contra Citomegalovirus/genética , Vacunas contra Citomegalovirus/inmunología , Femenino , Formaldehído , Infecciones por Herpesviridae/virología , Inmunización Secundaria , Inmunoglobulina G/sangre , Pulmón/virología , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/genética , Muromegalovirus/patogenicidad , Células 3T3 NIH , Pruebas de Neutralización , Vacunación , Vacunas de ADN/inmunología , Vacunas de Productos Inactivados/genética , Vacunas de Productos Inactivados/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
We previously demonstrated that immunization of mice with plasmid DNAs (pDNAs) expressing the murine cytomegalovirus (MCMV) genes IE1-pp89 and M84 provided synergistic protection against sublethal viral challenge, while immunization with plasmids expressing putative virion proteins provided no or inconsistent protection. In this report, we sought to augment protection by increasing the breadth of the immune response. We identified another MCMV gene (m04 encoding gp34) that provided strong and consistent protection against viral replication in the spleen. We also found that immunization with a DNA pool containing 10 MCMV genes that individually were nonprotective elicited reproducible protection against low to intermediate doses of challenge virus. Moreover, inclusion of these plasmids into a mixture with gp34, pp89, and M84 DNAs provided even greater protection than did coimmunization with pp89 and M84. The highest level of protection was achieved by immunization of mice with the pool of 13 pDNAs, followed by formalin-inactivated MCMV (FI-MCMV). Immunization with FI-MCMV elicited neutralizing antibodies against salivary gland-derived MCMV, and of greatest importance, mice immunized with both the combined pDNA pool and FI-MCMV had undetectable levels of virus in the spleen and salivary glands after challenge. Intracellular cytokine staining of splenocytes from pDNA- and FI-MCMV-immunized mice showed that pDNA immunization elicited high levels of pp89- and M83-specific CD8(+) T cells, whereas both pDNA and FI-MCMV immunizations generated strong CD8(+)-T-cell responses against virion-associated antigens. Taken together, these results show that immunization with pDNA and inactivated virus provides strong antibody and cell-mediated immunity against CMV infection.
Asunto(s)
Vacunas contra Citomegalovirus/administración & dosificación , Infecciones por Herpesviridae/prevención & control , Muromegalovirus , Vacunación , Vacunas de ADN/administración & dosificación , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos/inmunología , Proteínas Portadoras/genética , Vacunas contra Citomegalovirus/inmunología , Femenino , Glicoproteínas/genética , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/inmunología , Esquemas de Inmunización , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Muromegalovirus/genética , Muromegalovirus/inmunología , Muromegalovirus/fisiología , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Plásmidos/administración & dosificación , Vacunas de ADN/inmunología , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/inmunología , Proteínas Virales , Replicación ViralRESUMEN
We previously showed that intradermal immunization with plasmids expressing the murine cytomegalovirus (MCMV) protein IE1-pp89 or M84 protects against viral challenge and that coimmunization has a synergistic protective effect (C. S. Morello, L. D. Cranmer, and D. H. Spector, J. Virol. 74:3696-3708, 2000). Using an intracellular gamma interferon cytokine staining assay, we have now characterized the CD8+ T-cell response after DNA immunization with pp89, M84, or pp89 plus M84. The pp89- and M84-specific CD8+ T-cell responses peaked rapidly after three immunizations. DNA immunization and MCMV infection generated similar levels of pp89-specific CD8+ T cells. In contrast, a significantly higher level of M84-specific CD8+ T cells was elicited by DNA immunization than by MCMV infection. Fusion of ubiquitin to pp89 enhanced the CD8+ T-cell response only under conditions where vaccination was suboptimal. Three immunizations with either pp89, M84, or pp89 plus M84 DNA also provided significant protection against MCMV infection for at least 6 months, with the best protection produced by coimmunization. A substantial percentage of antigen-specific CD8+ T cells remained detectable, and they responded rapidly to the MCMV challenge. These results underscore the importance of considering antigens that do not appear to be highly immunogenic during infection as DNA vaccine candidates.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas contra Citomegalovirus/inmunología , Infecciones por Herpesviridae/prevención & control , Proteínas Inmediatas-Precoces/inmunología , Muromegalovirus/inmunología , Fosfoproteínas/inmunología , Proteínas de la Matriz Viral/inmunología , Células 3T3 , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Vacunas contra Citomegalovirus/administración & dosificación , ADN Viral/inmunología , Femenino , Infecciones por Herpesviridae/virología , Proteínas Inmediatas-Precoces/genética , Inmunización , Memoria Inmunológica , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/genética , Fosfoproteínas/genética , Plásmidos/genética , Plásmidos/inmunología , Ubiquitina/genética , Ubiquitina/inmunología , Ubiquitina/metabolismo , Proteínas de la Matriz Viral/genéticaRESUMEN
We previously demonstrated that after vaccination of BALB/c mice with DNA encoding murine cytomegalovirus (MCMV) IE1 or M84, a similar level of protection against MCMV infection was achieved. However, the percentage of antigen-specific CD8(+) T cells elicited by IE1 was higher than that by M84 as measured by intracellular cytokine staining when splenocytes were stimulated with an epitope peptide (M. Ye at al., J. Virol. 76:2100-2112, 2002). We show here that after DNA vaccination with M84, a higher percentage of M84-specific CD8(+) T cells was detected when splenocytes were stimulated with J774 cells expressing full-length M84. When the defined M84 epitope 297-305 was deleted, the mutant DNA vaccine was still protective against MCMV replication and induced strong M84-specific CD8(+)-T-cell responses. The M84 gene was subsequently subcloned into three fragments encoding overlapping protein fragments. When mice were immunized with each of the M84 subfragment DNAs, at least two additional protective CD8(+)-T-cell epitopes were detected. In contrast to strong responses after DNA vaccination, M84-specific CD8(+)-T-cell responses were poorly induced during MCMV infection. The weak M84-specific response after MCMV infection was not due to poor antigen presentation in antigen-presenting cells, since both J774 macrophages and primary peritoneal macrophages infected with MCMV in vitro were able to efficiently and constitutively present M84-specific epitopes starting at the early phase of infection. These results indicate that antigen presentation by macrophages is not sufficient for M84-specific CD8(+)-T-cell responses during MCMV infection.