RESUMEN
We developed a novel platform to express high levels of recombinant lipoproteins with intrinsic adjuvant properties. Based on this technology, our group developed recombinant lipidated dengue envelope protein domain IIIs as vaccine candidates against dengue virus. This work aims to evaluate the immune responses in mice to the tetravalent formulation. We demonstrate that 4 serotypes of recombinant lipidated dengue envelope protein domain III induced both humoral and cellular immunity against all 4 serotypes of dengue virus on the mixture that formed the tetravalent formulation. Importantly, the immune responses induced by the tetravalent formulation in the absence of the exogenous adjuvant were functional in clearing the 4 serotypes of dengue virus in vivo. We affirm that the tetravalent formulation of recombinant lipidated dengue envelope protein domain III is a potential vaccine candidate against dengue virus and suggest further detailed studies of this formulation in nonhuman primates.
Asunto(s)
Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Dengue/prevención & control , Lipoproteínas/inmunología , Proteínas Recombinantes/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Vacunas contra el Dengue/administración & dosificación , Vacunas contra el Dengue/genética , Modelos Animales de Enfermedad , Ensayo de Immunospot Ligado a Enzimas , Femenino , Inmunidad Celular , Inmunidad Humoral , Lipoproteínas/genética , Ratones Endogámicos BALB C , Pruebas de Neutralización , Dominios Proteicos , Proteínas Recombinantes/genética , Serogrupo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
The linkage of an immunogen with a toll-like receptor ligand has great potential to induce highly potent immune responses with the initial features of antigen-presenting cell activation. In the current study, we expressed recombinant dengue-3 envelope protein domain III (D3ED III) in lipidated form using an Escherichia coli-based system. The recombinant lipidated dengue-3 envelope protein domain III (LD3ED III) augments the expression levels of IL-12 family cytokines. LD3ED III-immunized mice enhance wide ranges of T cell responses as indicated by IFN-γ, IL-17, IL-21 production. Additionally, LD3ED III-immunized mice increase the frequencies of anti-D3ED III antibody producing cells. The boosted antibody titers cover various IgG isotypes, including IgG1, IgG2a, IgG2b, and IgG3. Importantly, LD3ED III-immunized mice induce neutralizing antibody capacity associated with a reduction of viremia levels after challenges. In contrast, mice that are immunized with D3ED III formulated with aluminum phosphate (D3ED III/Alum) only enhance Th2 responses and boost IgG1 antibody titers. Neither neutralizing antibody responses nor the inhibition of viremia levels after challenge is observed in mice that are immunized with D3ED III/Alum. These results suggest that LD3ED III can induce broad profiles of cellular and humoral immune responses.
Asunto(s)
Vacunas contra el Dengue/inmunología , Dengue/prevención & control , Proteínas del Envoltorio Viral/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Citocinas/inmunología , Células Dendríticas/inmunología , Virus del Dengue , Femenino , Inmunidad Celular , Inmunidad Humoral , Inmunoglobulina G/sangre , Lípidos/química , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/inmunología , Linfocitos T/inmunología , Viremia/prevención & controlRESUMEN
Synthetic lipopeptides have been widely used as vaccine adjuvants to enhance immune responses. The present study demonstrated that the tryptic N-terminal fragment of the lipoprotein rlipo-D1E3 (lipo-Nter) induces superior antitumor effects compared to a synthetic lipopeptide. The lipo-Nter was purified and formulated with protein or peptide vaccines to determine if lipo-Nter could be used as a novel adjuvant and could induce antitumor immunity in a cervical cancer model. Purified lipo-Nter activated the maturation of bone marrow-derived dendritic cells (BM-DCs), leading to the secretion of TNF-α through TLR2/6 but not TLR1/2. A recombinant mutant HPV16 E7 (rE7m) protein was mixed with lipo-Nter to immunize the mice; the anti-E7 antibody titers were increased, and the T helper cells were skewed toward the Th1 fate (increased IL-2 and decreased IL-5 secretion). Single-dose injection of rE7m and lipo-Nter inhibited tumor growth, but the injection of rE7m alone did not. Accordingly, lipo-Nter also enhanced the antitumor immunity of the E7-derived peptide but not the synthetic lipopeptide (Pam3CSK4). We demonstrated that the lipo-Nter of a bacterial-derived recombinant lipoprotein is a novel adjuvant that could be used for the development of a new generation of vaccines.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Lipopéptidos/inmunología , Lipopéptidos/farmacología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Neoplasias del Cuello Uterino/inmunología , Animales , Línea Celular Tumoral , Células Dendríticas/inmunología , Femenino , Inmunidad Celular/inmunología , Inmunización/métodos , Inmunoterapia/métodos , Interleucina-2/inmunología , Interleucina-5/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas E7 de Papillomavirus/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Neoplasias del Cuello Uterino/terapia , Vacunas de Subunidad/inmunologíaRESUMEN
The combination of recombinant protein antigens with an immunostimulator has the potential to greatly increase the immunogenicity of recombinant protein antigens. In the present study, we selected the dengue-4 envelope protein domain III as a dengue vaccine candidate and expressed the protein in lipidated form using an Escherichia coli-based system. The recombinant lipidated dengue-4 envelope protein domain III folded into the proper conformation and competed with the dengue-4 virus for cellular binding sites. Mice immunized with lipidated dengue-4 envelope protein domain III without exogenous adjuvant had higher frequencies of dengue-4 envelope protein domain III-specific B cells secreting antibodies than mice immunized with the nonlipidated form. Importantly, lipidated dengue-4 envelope protein domain III-immunized mice demonstrated a durable neutralizing antibody response and had reduced viremia levels after challenge. The study demonstrates that lipidated dengue-4 envelope protein domain III is immunogenic and may be a potential dengue vaccine candidate. Furthermore, the lipidation strategy can be applied to other serotypes of dengue virus.
Asunto(s)
Vacunas contra el Dengue/inmunología , Dengue/prevención & control , Lípidos/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Línea Celular , Cricetinae , Femenino , Inmunidad Humoral , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/inmunología , Viremia/prevención & controlRESUMEN
BACKGROUND: Dengue virus is a mosquito-transmitted virus that can cause self-limiting dengue fever, severe life-threatening dengue hemorrhagic fever and dengue shock syndrome. The existence of four serotypes of dengue virus has complicated the development of an effective and safe dengue vaccine. Recently, a clinical phase 2b trial of Sanofi Pasteur's CYD tetravalent dengue vaccine revealed that the vaccine did not confer full protection against dengue-2 virus. New approaches to dengue vaccine development are urgently needed. Our approach represents a promising method of dengue vaccine development and may even complement the deficiencies of the CYD tetravalent dengue vaccine. METHODOLOGY/PRINCIPAL FINDINGS: Two important components of a vaccine, the immunogen and immunopotentiator, were combined into a single construct to generate a new generation of vaccines. We selected dengue-2 envelope protein domain III (D2ED III) as the immunogen and expressed this protein in lipidated form in Escherichia coli, yielding an immunogen with intrinsic immunopotentiation activity. The formulation containing lipidated D2ED III (LD2ED III) in the absence of exogenous adjuvant elicited higher D2ED III-specific antibody responses than those obtained from its nonlipidated counterpart, D2ED III, and dengue-2 virus. In addition, the avidity and neutralizing capacity of the antibodies induced by LD2ED III were higher than those elicited by D2ED III and dengue-2 virus. Importantly, we showed that after lipidation, the subunit candidate LD2ED III exhibited increased immunogenicity while reducing the potential risk of antibody-dependent enhancement of infection in mice. CONCLUSIONS/SIGNIFICANCE: Our study suggests that the lipidated subunit vaccine approach could be applied to other serotypes of dengue virus and other pathogens.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Acrecentamiento Dependiente de Anticuerpo , Vacunas contra el Dengue/inmunología , Dengue/prevención & control , Proteínas del Envoltorio Viral/inmunología , Animales , Dengue/inmunología , Vacunas contra el Dengue/administración & dosificación , Escherichia coli/genética , Ratones , Ratones Endogámicos BALB C , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
Many attempts have focused on the use of either immunomodulators or antigen delivery systems to obtain an efficacious vaccine. Here, we report a novel approach that combined an immunomodulator and delivery system to enhance antigen association and induce robust immunity. We expressed a recombinant lipidated dengue-1 envelope protein domain III (LD1ED III) and its non-lipidated form, D1ED III, in an Escherichia coli system. The LD1ED III contains a bacterial lipid moiety, which is a potent immunomodulator. We demonstrated that LD1ED III possesses an inherent immunostimulation ability that can activate RAW 264.7 macrophage cells by up-regulating their expression of CD40, CD80, CD83, CD86 and MHC II, whereas D1ED III could not induce the up-regulation of these molecules. Moreover, combining LD1ED III with a multiphase emulsion system (called PELC) increased the antigen association more than either combining D1ED III with PELC or the antigen alone. Enhanced antigen association has been shown to correlate with stronger T cell responses, greater antibody avidity and improved neutralizing capacity. Our results demonstrate that combining recombinant lipoproteins with PELC improved both the intensity and the quality of the immune response. This approach is a promising strategy for the development of subunit vaccines that induce robust immunity.
Asunto(s)
Vacunas contra el Dengue/administración & dosificación , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Emulsiones/administración & dosificación , Lípidos/administración & dosificación , Proteínas del Envoltorio Viral/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales/sangre , Afinidad de Anticuerpos , Antígenos CD/biosíntesis , Línea Celular , Vacunas contra el Dengue/genética , Virus del Dengue/genética , Escherichia coli/genética , Expresión Génica , Antígenos de Histocompatibilidad Clase II/biosíntesis , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Ratones Endogámicos BALB C , Linfocitos T/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
We have previously demonstrated that vaccination with a subunit dengue vaccine containing a consensus envelope domain III with aluminum phosphate elicits neutralizing antibodies against all four serotypes of dengue virus in mice. In this study, we evaluated the immunogenicity of the subunit dengue vaccine in non-human primates. After vaccination, monkeys that received the subunit vaccine with aluminum phosphate developed a significantly strong and long-lasting antibody response. A specific T cell response with cytokine production was also induced, and this correlated with the antibody response. Additionally, neutralizing antibodies against serotype 2 were detected in two of three monkeys. The increase in serotype-2-specific antibody titers and avidity observed in these two monkeys suggested that a serotype-2-biased antibody response occurs. These data provide evidence that a protective neutralizing antibody response was successfully elicited in non-human primates by the dengue subunit vaccine with aluminum phosphate adjuvant.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Proteínas del Envoltorio Viral/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Aluminio/administración & dosificación , Animales , Afinidad de Anticuerpos , Citocinas/metabolismo , Vacunas contra el Dengue/administración & dosificación , Vacunas contra el Dengue/genética , Virus del Dengue/genética , Haplorrinos , Fosfatos/administración & dosificación , Linfocitos T/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Proteínas del Envoltorio Viral/genéticaRESUMEN
The major weaknesses of subunit vaccines are their low immunogenicity and poor efficacy. Adjuvants can help to overcome some of these inherent defects with subunit vaccines. Here, we evaluated the efficacy of the newly developed water-in-oil-in-water multiphase emulsion system, termed PELC, in potentiating the protective capacity of dengue-1 envelope protein domain III. Unlike aluminum phosphate, dengue-1 envelope protein domain III formulated with PELC plus CpG oligodeoxynucleotides induced neutralizing antibodies against dengue-1 virus and increased the splenocyte secretion of IFN-γ after in vitro re-stimulation. The induced antibodies contained both the IgG1 and IgG2a subclasses. A rapid anamnestic neutralizing antibody response against a live dengue virus challenge was elicited at week 26 after the first immunization. These results demonstrate that PELC plus CpG oligodeoxynucleotides broaden the dengue-1 envelope protein domain III-specific immune responses. PELC plus CpG oligodeoxynucleotides is a promising adjuvant for recombinant protein based vaccination against dengue virus.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Vacunas contra el Dengue/administración & dosificación , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Aceites/administración & dosificación , Oligodesoxirribonucleótidos/administración & dosificación , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Vacunas contra el Dengue/genética , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos BALB C , Bazo/inmunología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
To protect against dengue viral infection, a novel lipidated dengue subunit vaccine was rationally designed to contain the consensus amino acid sequences derived from four serotypes of dengue viruses. We found that the lipidated consensus dengue virus envelope protein domain III (LcED III) is capable of activating antigen-presenting cells and enhancing cellular and humoral immune responses. A single-dose of LcED III immunization in mice without extra adjuvant formulation is sufficient to elicit neutralizing antibodies against all four serotypes of dengue viruses. In addition, strong memory responses were elicited in mice immunized with a single-dose of LcED III. Quick, anamnestic neutralizing antibody responses to a live dengue virus challenge were elicited at week 28 post-immunization. These results demonstrate the promising possibility of a future successful tetravalent vaccine against dengue viral infections that utilizes one-dose vaccination with LcED III.
Asunto(s)
Vacunas contra el Dengue/inmunología , Memoria Inmunológica/inmunología , Animales , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Células K562 , Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB CRESUMEN
The structural analysis of post-translational modifications (PTMs) of lipoproteins is difficult due to the hydrophobic properties of their fatty acid moieties. At the present time, the relative positions of fatty acid components on the N-acyl-S-diacylglycerylcysteine core structure has not been specifically identified in any natural or bacterial expressed recombinant lipoproteins. In this study, we describe a rapid solid-phase extraction using acetonitrile and isopropanol method that can be performed manually to isolate large amounts of relatively pure lipopeptides generated by the limited tryptic-digestion of recombinant lipoproteins. Using these lipopeptides and LC/MS mass spectra analysis, two groups of N-terminal lipidated (diacyl or triacyl) molecules that differ by one fatty acid unit were successfully identified. This LC/MS method also provided the separation of lipopeptides differing by 14 Da for the on-line MS identification. Multiple-stage fragmentation analyses of the di- and triacyl lipopeptides using both the positive and negative ion modes enabled to identify the putative structure of the N-acyl-S-diacylglycerylcysteine containing an amide bond to palmitic acid at the N-terminal cysteine, a palmitic acid at sn1 position, and an unsaturated fatty acid of either hexadecenoic acid, cyclopropaneoctanoic acid, oleic acid and nonadecenoic acid at sn2 position of diacylglycerol residue through ester bonding. For diacyl lipoprotein, the saturated palmitoyl fatty acid group is absent at sn1 position of glycerol-derived lipid residue of lipopeptide.
Asunto(s)
Bacterias/química , Cromatografía Líquida de Alta Presión/métodos , Lipopéptidos/química , Lipopéptidos/aislamiento & purificación , Lipoproteínas/química , Espectrometría de Masas/métodos , Lipoproteínas/genética , Estructura Molecular , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
The lipid moiety of a novel recombinant lipoprotein, which contains a dengue virus envelope protein domain 3, rlipo-D1E3, has been shown to activate antigen-presenting cells (APCs) as an intrinsic adjuvant. Because the lipid moiety of rlipo-D1E3 contains an unsaturated fatty acid, it is unclear if the receptor usage by bacterially derived lipoproteins is the same as that of the synthetic lipopeptide palmitoyl-3-Cys-Ser-(Lys)(4) (Pam3). In the present study, we show that the rlipo-D1E3 lipoprotein can induce the activation of spleen cells and bone marrow-derived dendritic cells (BM-DCs) in wild-type and TLR4-deficient mice, but not in TLR2(-/-) mice. After analyzing the co-receptor usage of TLR2 using TLR1(-/-) or TLR6(-/-) mice, the TLR2 signaling triggered by rlipo-D1E3 and Pam3 could use either TLR1 or TLR6 as a co-receptor. Analysis of the MAPK signaling pathway revealed that rlipo-D1E3 could initiate the phosphorylation of p38, ERK1/2 and JNK1/2 earlier than the synthetic lipopeptide. In addition, the expression levels of IL-23, IL-27 and MIP-1 alpha in BM-DCs stimulated by rlipo-D1E3 were higher than the expression levels in BM-DCs stimulated by Pam3. Taken together, these results demonstrate that different TLR2 ligands can promote various immune responses by inducing different levels of biological cytokines and chemokines.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Perfilación de la Expresión Génica , Lipoproteínas/farmacología , FN-kappa B/fisiología , Transducción de Señal/fisiología , Receptor Toll-Like 2/fisiología , Animales , Quimiocinas/genética , Citocinas/biosíntesis , Citocinas/genética , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Proteínas Recombinantes/farmacologíaRESUMEN
We have developed a novel platform technology that can express high levels of recombinant lipoproteins with intrinsic adjuvant properties. In this study, Ag473 (a lipoprotein from Neisseria meningitidis) can be produced in high yields using Escherichia coli strain C43 (DE3). After testing a non-lipoimmunogen (E3, from dengue virus) fused with different lipid signal peptides from other lipoproteins as well as Ag473 fragments of different lengths, we identified that the fusion sequence has to contain at least the N-terminal 40 residues, D1, of Ag473 to achieve high expression levels of the recombinant lipo-immunogen (rlipo-D1E3). The rlipo-D1E3 was found to elicit stronger anti-E3 and virus neutralizing antibody responses in animal studies than those from rE3 alone or rE3 formulated with alum adjuvant. These results have successfully demonstrated the merit of lipo-immunogens for novel vaccine development.
Asunto(s)
Proteínas Bacterianas/inmunología , Lipoproteínas/inmunología , Vacunas Sintéticas/inmunología , Vacunas/biosíntesis , Secuencia de Aminoácidos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Diseño de Fármacos , Escherichia coli/genética , Lipoproteínas/biosíntesis , Datos de Secuencia Molecular , Pruebas de Neutralización , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/biosíntesisRESUMEN
A novel dengue vaccine candidate comprised of a consensus dengue virus envelope protein domain III (cED III) was developed to fight against dengue virus infection. The amino acid sequence of this novel cED III was obtained by alignment of amino acid sequences from different isolates of the four serotypes of dengue viruses. A proof-of-concept study demonstrated that BALB/c mice immunized with the recombinant cED III developed neutralizing antibodies against all serotypes of dengue virus. Moreover, formulation of recombinant cED III with aluminum phosphate could induce long-lasting antibody responses and anamnestic neutralizing antibody responses following challenge with dengue virus at week 28 after priming. These results demonstrate the possibility of developing a single tetravalent vaccine against dengue viral infections.