A successful vaccine depends on its capacity to elicit a protective immune response against the t... more A successful vaccine depends on its capacity to elicit a protective immune response against the target pathogen. The adjuvant used plays an important role in enhancing and directing the immune response. Liposomes are vaccine adjuvants that allow the co-encapsulation of antigens and immunostimulants. Our aim was to evaluate the adjuvanticity of a cationic liposome (Lip) formulated with a novel gemini lipopeptide (AG2-C16) alone or in combination with CpG-ODN as immunostimulants. To achieve this, we used the recombinant clumping factor of Staphylococcus aureus (rClfA) as a model antigen, in a murine model. We characterized the formulations by DLS, Cryo-SEM, and TEM, and analyzed the humoral and cellular immune responses induced in BALB/c and C57BL/6J mice injected with free rClfA and three formulations: Lip + CpG-ODN + rClfA, Lip + AG2-C16 + rClfA and Lip + AG2-C16 + CpG-ODN + rClfA. The addition of immunostimulants to the liposomes did not change the membrane diameter but affected their hydrodynamic diameter, z-potential, and homogeneity. All liposomal formulations were able to stimulate a specific humoral response, with high serum IgG, IgG1 and IgG2a or IgG2c titers in BALB/ c or C57BL/6J mice, respectively. In addition, increased vaginal IgG levels were detected after injection, with no specific IgA. The cellular immunity induced by Lip + AG2-C16 + CpG-ODN + rClfA was characterized by a predominant Th1 profile, with the co-induction of Th2 and Th17 cells, and IFN-γ + cytotoxic T cells. Furthermore, we studied the capacity of the different formulations to stimulate murine keratinocytes and fibroblasts in vitro. While no formulation activated keratinocytes, Lip + AG2-C16 + CpG-ODN increased the expression of CXCL9 in fibroblasts. These results suggest Lip + AG2-C16 + CpG-ODN as a promising adjuvant candidate to be used in vaccines against pathogens that require Th1/Th2/Th17 combined profiles, like S. aureus. Additionally, based on the IFN-γ + cytotoxic T cells stimulation and the CXCL9 production by fibroblasts, we propose the use of this adjuvant formulation for the stimulation of a Th1 profile.
Journal of tissue engineering and regenerative medicine, Jan 6, 2017
The development of three-dimensional models of reconstituted mouse epidermis (RME) has been hampe... more The development of three-dimensional models of reconstituted mouse epidermis (RME) has been hampered by the difficulty to maintain murine primary keratinocyte cultures and to achieve a complete epidermal stratification. In this study, a new protocol is proposed for the rapid and convenient generation of RME, which reproduces accurately the architecture of a normal mouse epidermis. During RME morphogenesis, the expression of differentiation markers such as keratins, loricrin, filaggrin, E-cadherin and connexins was followed, showing that RME structure at day 5 was similar to those of a normal mouse epidermis, with the acquisition of the natural barrier function. It was also demonstrated that RME responded to skin-relevant proinflammatory cytokines by increasing the expression of antimicrobial peptides and chemokines, and inhibiting epidermal differentiation markers, as in the human system. This new model of RME is therefore suitable to investigate mouse epidermis physiology further a...
The control of the worldwide spread of sexually transmitted Chlamydia trachomatis (Ct) infection ... more The control of the worldwide spread of sexually transmitted Chlamydia trachomatis (Ct) infection urgently demands the development of a preventive vaccine. In this work, we designed a vaccine based on a fragment of polymorphic protein D (FPmpD) that proved to be immunogenic enough to generate a robust systemic and mucosal IgG humoral immune response in two strains of mice. We used a heterologous prime-boost strategy, including simultaneous systemic and mucosal administration routes. The high titers of anti-PmpD antibodies elicited by this immunization scheme did not affect murine fertility. We tested the vaccine in a mouse model of Ct intravaginal infection. Anti-PmpD antibodies displayed potent neutralizing activity in vitro and protective effects in uterine tissues in vivo. Notably, the humoral immune response of PmpD-vaccinated mice was faster and stronger than the primary immune response of non-vaccinated mice when exposed to Ct. FPmpD-based vaccine effectively reduced Ct sheddin...
The aims of the present study were to evaluate the ability of a subunit vaccine composed of recom... more The aims of the present study were to evaluate the ability of a subunit vaccine composed of recombinant molecules of α-toxin, β-toxin, FnBPA and ClfA, formulated with cationic liposomes and CpG-ODN, to confer protection against natural S. aureus intramammary infection (IMI) and to assess the antibody response against the vaccine components. A stringent criterion based on molecular identification of the isolates was used to define IMI. The proportion of animals that developed new S. aureus IMI was higher in the Control group compared with the Vaccine group (reduction of 60.7%), and time to new S. aureus IMI was higher for animals in the Vaccine group compared with animals in the Control group, although not statistically significant. Molecular identification of the isolates allowed the detection of S. aureus pulsotypes that appeared transiently in milk and others that were able to establish IMI, providing a new perspective to define parameters related to the definition of new IMI and cures. Specific IgG, IgG1 and IgG2 levels against the four recombinant proteins included in the vaccine were significantly increased in the vaccinated group and the recombinant α-toxin included in the vaccine generated antibodies that reduced significantly the haemolytic activity of native α-toxin. Data reported in the present study indicate a possible effect on both the proportion of animals developing new IMI and the time to new S. aureus IMI, but the incidence of disease within the study was too low to provide statistical confirmation.
The development of novel vaccines requires the design of new adjuvants able to give long lastingi... more The development of novel vaccines requires the design of new adjuvants able to give long lastingimmune responses. Our aim was to obtain cationic liposomes as adjuvants by an industry-suitablemethod, and evaluate them using bovine serum albumin (BSA) as immunogen and CpGoligonucleotides with phosphodiesther bonds, as immunostimulants. Liposomes (Lip) wereprepared with dipalmitoylphosphatidylcholine, cholesterol and stearylamine by Ethanol Injectionmethod. Immune response was assessed by immunization of Balb/c mice with: Lip+BSALip+BSA+CpG, CpG+BSA or aluminium hydroxide (Al(OH)3+BSA). Liposomal formulations wereable to induce high antibody levels. Lip+BSA+CpG led to higher IgG levels than Lip+BSA (p<0.05,Mann-Whitney) and elicited the highest IgG2a levels. All the formulations induced antigen specificcellular proliferation, without significant differences, meanwhile Lip+BSA+CpG produced thehighest levels of IFN-γ. These results showed these liposomes are versatile vehicles to pote...
A successful vaccine depends on its capacity to elicit a protective immune response against the t... more A successful vaccine depends on its capacity to elicit a protective immune response against the target pathogen. The adjuvant used plays an important role in enhancing and directing the immune response. Liposomes are vaccine adjuvants that allow the co-encapsulation of antigens and immunostimulants. Our aim was to evaluate the adjuvanticity of a cationic liposome (Lip) formulated with a novel gemini lipopeptide (AG2-C16) alone or in combination with CpG-ODN as immunostimulants. To achieve this, we used the recombinant clumping factor of Staphylococcus aureus (rClfA) as a model antigen, in a murine model. We characterized the formulations by DLS, Cryo-SEM, and TEM, and analyzed the humoral and cellular immune responses induced in BALB/c and C57BL/6J mice injected with free rClfA and three formulations: Lip + CpG-ODN + rClfA, Lip + AG2-C16 + rClfA and Lip + AG2-C16 + CpG-ODN + rClfA. The addition of immunostimulants to the liposomes did not change the membrane diameter but affected their hydrodynamic diameter, z-potential, and homogeneity. All liposomal formulations were able to stimulate a specific humoral response, with high serum IgG, IgG1 and IgG2a or IgG2c titers in BALB/ c or C57BL/6J mice, respectively. In addition, increased vaginal IgG levels were detected after injection, with no specific IgA. The cellular immunity induced by Lip + AG2-C16 + CpG-ODN + rClfA was characterized by a predominant Th1 profile, with the co-induction of Th2 and Th17 cells, and IFN-γ + cytotoxic T cells. Furthermore, we studied the capacity of the different formulations to stimulate murine keratinocytes and fibroblasts in vitro. While no formulation activated keratinocytes, Lip + AG2-C16 + CpG-ODN increased the expression of CXCL9 in fibroblasts. These results suggest Lip + AG2-C16 + CpG-ODN as a promising adjuvant candidate to be used in vaccines against pathogens that require Th1/Th2/Th17 combined profiles, like S. aureus. Additionally, based on the IFN-γ + cytotoxic T cells stimulation and the CXCL9 production by fibroblasts, we propose the use of this adjuvant formulation for the stimulation of a Th1 profile.
Journal of tissue engineering and regenerative medicine, Jan 6, 2017
The development of three-dimensional models of reconstituted mouse epidermis (RME) has been hampe... more The development of three-dimensional models of reconstituted mouse epidermis (RME) has been hampered by the difficulty to maintain murine primary keratinocyte cultures and to achieve a complete epidermal stratification. In this study, a new protocol is proposed for the rapid and convenient generation of RME, which reproduces accurately the architecture of a normal mouse epidermis. During RME morphogenesis, the expression of differentiation markers such as keratins, loricrin, filaggrin, E-cadherin and connexins was followed, showing that RME structure at day 5 was similar to those of a normal mouse epidermis, with the acquisition of the natural barrier function. It was also demonstrated that RME responded to skin-relevant proinflammatory cytokines by increasing the expression of antimicrobial peptides and chemokines, and inhibiting epidermal differentiation markers, as in the human system. This new model of RME is therefore suitable to investigate mouse epidermis physiology further a...
The control of the worldwide spread of sexually transmitted Chlamydia trachomatis (Ct) infection ... more The control of the worldwide spread of sexually transmitted Chlamydia trachomatis (Ct) infection urgently demands the development of a preventive vaccine. In this work, we designed a vaccine based on a fragment of polymorphic protein D (FPmpD) that proved to be immunogenic enough to generate a robust systemic and mucosal IgG humoral immune response in two strains of mice. We used a heterologous prime-boost strategy, including simultaneous systemic and mucosal administration routes. The high titers of anti-PmpD antibodies elicited by this immunization scheme did not affect murine fertility. We tested the vaccine in a mouse model of Ct intravaginal infection. Anti-PmpD antibodies displayed potent neutralizing activity in vitro and protective effects in uterine tissues in vivo. Notably, the humoral immune response of PmpD-vaccinated mice was faster and stronger than the primary immune response of non-vaccinated mice when exposed to Ct. FPmpD-based vaccine effectively reduced Ct sheddin...
The aims of the present study were to evaluate the ability of a subunit vaccine composed of recom... more The aims of the present study were to evaluate the ability of a subunit vaccine composed of recombinant molecules of α-toxin, β-toxin, FnBPA and ClfA, formulated with cationic liposomes and CpG-ODN, to confer protection against natural S. aureus intramammary infection (IMI) and to assess the antibody response against the vaccine components. A stringent criterion based on molecular identification of the isolates was used to define IMI. The proportion of animals that developed new S. aureus IMI was higher in the Control group compared with the Vaccine group (reduction of 60.7%), and time to new S. aureus IMI was higher for animals in the Vaccine group compared with animals in the Control group, although not statistically significant. Molecular identification of the isolates allowed the detection of S. aureus pulsotypes that appeared transiently in milk and others that were able to establish IMI, providing a new perspective to define parameters related to the definition of new IMI and cures. Specific IgG, IgG1 and IgG2 levels against the four recombinant proteins included in the vaccine were significantly increased in the vaccinated group and the recombinant α-toxin included in the vaccine generated antibodies that reduced significantly the haemolytic activity of native α-toxin. Data reported in the present study indicate a possible effect on both the proportion of animals developing new IMI and the time to new S. aureus IMI, but the incidence of disease within the study was too low to provide statistical confirmation.
The development of novel vaccines requires the design of new adjuvants able to give long lastingi... more The development of novel vaccines requires the design of new adjuvants able to give long lastingimmune responses. Our aim was to obtain cationic liposomes as adjuvants by an industry-suitablemethod, and evaluate them using bovine serum albumin (BSA) as immunogen and CpGoligonucleotides with phosphodiesther bonds, as immunostimulants. Liposomes (Lip) wereprepared with dipalmitoylphosphatidylcholine, cholesterol and stearylamine by Ethanol Injectionmethod. Immune response was assessed by immunization of Balb/c mice with: Lip+BSALip+BSA+CpG, CpG+BSA or aluminium hydroxide (Al(OH)3+BSA). Liposomal formulations wereable to induce high antibody levels. Lip+BSA+CpG led to higher IgG levels than Lip+BSA (p<0.05,Mann-Whitney) and elicited the highest IgG2a levels. All the formulations induced antigen specificcellular proliferation, without significant differences, meanwhile Lip+BSA+CpG produced thehighest levels of IFN-γ. These results showed these liposomes are versatile vehicles to pote...
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