Liposomal co-encapsulation of a novel gemini lipopeptide and a CpG-ODN induces a strong Th1 response with the co-activation of a Th2/Th17 profile and high antibody levels.

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.

Evaluation of three formulations based on Polymorphic membrane protein D in mice infected with Chlamydia trachomatis.

The significant impact of Chlamydia trachomatis(Ct) infections worldwide highlights the need to develop a prophylactic vaccine that elicits effective immunity and protects the host from the immunopathological effects of Ct infection. The aim of this study was to evaluate a vaccine based on a fragment of the Polymorphic membrane protein D (FPmpD) of C. trachomatis as an immunogen using a heterologous DNA prime-protein boost strategy in female mice Three different formulations were evaluated as protein boost: free recombinant FPmpD (rFPmpD) or rFPmpD formulated with a liposomal adjuvant alternatively supplemented with CpG or a cationic gemini lipopeptide as immunostimulants. The three candidates induced an increase in the cervicovaginal and systemic titers of anti-rFPmpD antibodies in two strains of mice (BALB/c and C57BL/6), with no evidence of fertility alterations. The three formulations induced a rapid and robust humoral immune response upon the Ct challenge. However, the booster with free rFPmpD more efficiently reduced the shedding of infective Ct and prevented the development of immunopathology. The formulations containing adjuvant induced a strong inflammatory reaction in the uterine tissue. Hence, the prime-boost strategy with the adjuvant-free FPmpD vaccine formulation might constitute a promissory candidate to prevent C. trachomatis intravaginal infection.

Heterologous prime-boost vaccination based on Polymorphic protein D protects against intravaginal Chlamydia trachomatis infection in mice.

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 shedding into cervicovaginal fluids, bacterial burden at the genitourinary tract, and overall infectivity. Hence, the FPmpD-based vaccine might constitute an efficient tool to protect against Ct intravaginal infection and decrease the infection spreading.

Neospora caninum truncated recombinant proteins formulated with liposomes and CpG-ODNs triggered a humoral immune response in cattle after immunisation and challenge.

Abortions caused by Neospora caninum are a serious problem in cattle production and require effective immunoprophylaxis. The objective of this work was to assess the humoral immune response to four recombinant (r) N. caninum antigens in cattle after immunisation and challenge. MIC1 and MIC3 proteins from the micronemes, SRS2 from the surface of tachyzoites, and GRA7 from the dense granules were expressed as truncated recombinant proteins in Escherichia coli. Cationic liposomes (Lip) and CpG oligodeoxynucleotides (CpG-ODNs) were used as adjuvant. Steers were assigned to three groups of six steers each and were inoculated twice subcutaneously, 21 days apart. The rP + Lip + CpG-ODN group received the truncated recombinant proteins rMIC1, rMIC3, rSRS2 and rGRA7 formulated with the adjuvant; the Lip + CpG-ODN group received the adjuvant alone; and the PBS group received sterile phosphate-buffered saline. All steers were subcutaneously challenged with the NC-1 strain of N. caninum 35 days after the second dose of immunisation. Steers from the rP + Lip + CpG-ODN group developed specific IgG, IgG1 and IgG2 against the four recombinant proteins after immunisation. After challenge, IgG against rMIC1 and rMIC3 was detected in rP + Lip + CpG-ODN group and against rSRS2 and rGRA7 in all groups. IgG1 and IgG2 against the four recombinant proteins remained high after challenge in the rP + Lip + CpG-ODN group. Indirect ELISA detected anti-N. caninum antibodies after challenge in all groups, with the highest level of antibodies being detected in the rP + Lip + CpG-ODN group. The recombinant vaccine formulated with rMIC1, rMIC3, rSRS2 and rGRA7 using Lip + CpG-ODN as adjuvant was immunogenic in cattle and the humoral immune response after challenge was enhanced in vaccinated cattle.

Gemini lipopeptides as vaccine adjuvants: a new role for these versatile carriers.

The design of subunit vaccines requires new adjuvant systems. We designed and synthesized new lipopeptides (cysteine-based) of low molecular weight with different hydrophobic chains that dimerize becoming gemini lipopeptides. They were characterized and their adjuvant capacity was tested in mice by the inoculation of a protein antigen formulated with the lipopeptides, with and without the addition of CpG-oligodeoxynucleotides. Formulations were able to induce an immune response and produced no adverse effects. An adjuvant ability is described for the first time for this type of molecules.

Liposomal and CpG-ODN formulation elicits strong humoral immune responses to recombinant Staphylococcus aureus antigens in heifer calves.

Bovine mastitis caused by Staphylococcus aureus is a serious problem in dairy production and effective immunoprophylaxis is an unmet goal so far. The objective of this work was to assess the humoral immune response of heifer calves against two recombinant S. aureus antigens: Clumping factor A (ClfA) and Fibronectin Binding Protein A (FnBPA), formulated with a novel adjuvant based on cationic liposomes (Lip) and CpG oligodeoxynucleotides (CpG-ODN). Six groups of 6-8 months old heifer calves received three doses biweekly of antigens, formulated with Al(OH)3, liposomes, CpG-ODN or Lip + CpG-ODN. Animals also received a fourth dose after a year (day 410) and a booster before calving. The administration of Al(OH)3+FnBPA/ClfA and Lip + FnBPA/ClfA + CpG-ODN induced the highest specific IgG levels, after the first 3 doses and induced a fast increase of antibodies after the fourth dose. All the formulations stimulated the production of specific IgG1, after the third and fourth dose. Specific IgG2 for both proteins was only stimulated after the fourth dose by Lip + FnBPA/ClfA + CpG-ODN. Pre-calving immunisation with Lip + FnBPA/ClfA + CpG-ODN led to the highest IgG levels during the calving period and to the production of the IgG2 subclass. The formulation was also able to stimulate the highest antibody levels in milk, 30 and 45 days after pre-calving booster. The combination of liposomes and CpG-ODN as adjuvant for a subunit vaccine, together with the immunisation schedule described, induced a strong humoral immune response with production of specific IgG2. The formulation demonstrated to induce immune memory allowing the application of a single pre-calving booster to maintain high antibody levels throughout the period of increased susceptibility to intramammary infections.