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.

Validation of an indirect in-house ELISA using synthetic peptides to detect antibodies anti-gp90 and gp45 of the equine infectious anaemia virus.

BACKGROUND: Equine infectious anaemia (EIA) is controlled by the identification of seropositive animals. The official diagnostic method is the agar gel immunodiffusion (AGID) test, which detects antibodies against a viral core protein (p26). Although AGID is inexpensive and specific, the report of results takes considerable time and the test has low analytical sensitivity. OBJECTIVE: To validate our in-house indirect ELISAgp90/45 , following the World Organization of Animal Health (OIE) criteria. STUDY DESIGN: Test validation. METHODS: Synthetic peptides gp90 and gp45 were used as antigens in ELISAgp90/45 . Tests used for validation, calibration and linear working operating range, analytical and diagnostic sensitivity and specificity, repeatability and reproducibility were assessed by comparing them with the AGID test and using 1844 equine sera grouped into five different panels. RESULTS: We were able to replace the National References Sera with our Internal Reference Sera. ELISAgp90/45 had acceptable repeatability and reproducibility. Analytical sensitivity of the ELISAgp90/45 was 800 times greater than that of AGID test for positive sera and 400 times greater for weak positive sera. ELISAgp90/45 also showed optimal analytical specificity, since no cross-reactivity was detected with antibodies against other equine viruses. One sample was positive by AGID test and negative by ELISAgp90/45. ELISAgp90/45 was performed using 243 EIA positive and 878 negative equid sera, and showed a diagnostic sensitivity of 99.59% [CI 97.73%-99.99%] and a diagnostic specificity of 90.32% [CI 88.17%-92.19%], compared to AGID test; thus, it was demonstrated to be a robust test. MAIN LIMITATIONS: Samples were derived from naturally infected equid populations showing heterogeneous clinical states: therefore, their status was uncertain and some horses were sampled more than once. The AGID test may not be the most useful gold standard. CONCLUSION: ELISAgp90/45 is a useful tool for the diagnosis of EIAV infection and meets validation requirements established by the OIE.

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.

In silico prediction of T- and B-cell epitopes in PmpD: First step towards to the design of a Chlamydia trachomatis vaccine.

BACKGROUND: Chlamydia trachomatis is the most common sexually transmitted bacterial infection globally. Currently, there are no vaccines available despite the efforts made to develop a protective one. Polymorphic membrane protein D (PmpD) is an attractive immunogen candidate as it is conserved among strains and it is target of neutralizing antibodies. However, its high molecular weight and its complex structure make it difficult to handle by recombinant DNA techniques. Our aim is to predict B-cell and T-cell epitopes of PmpD. METHOD: A sequence (Genbank AAK69391.2) having 99-100% identity with various serovars of C. trachomatis was used for predictions. NetMHC and NetMHCII were used for T-cell epitope linked to MHC I or MHC II alleles prediction, respectively. BepiPred predicted linear B-cell epitopes. For three dimensional epitopes, PmpD was homology-modeled by Raptor X. Surface epitopes were predicted on its globular structure using DiscoTope. RESULTS: NetMHC predicted 271 T-cell epitopes of 9-12aa with weak affinity, and 70 with strong affinity to MHC I molecules. NetMHCII predicted 2903 T-cell epitopes of 15aa with weak affinity, and 742 with strong affinity to MHC II molecules. Twenty four linear B-cell epitopes were predicted. Raptor X was able to model 91% of the three-dimensional structure whereas 57 residues of discontinuous epitopes were suggested by DiscoTope. Six regions containing B-cell and T-cell epitopes were identified by at least two predictors. CONCLUSIONS: PmpD has potential B-cell and T-cell epitopes distributed throughout the sequence. Thus, several fragments were identified as valuable candidates for subunit vaccines against C. trachomatis.

Immune mediators associated to male infertility in a mouse model of DNA immunization with the sperm protease proacrosin.

The immune response has relevant physiological functions both in the male and female reproductive system, and must be tightly controlled to achieve a successful pregnancy. Several immune factors have been related to infertility, among them humoral and cellular immune responses triggered by sperm antigens. The present study was aimed at evaluating the immune profile induced by DNA immunization against the sperm protease proacrosin in CF1 male mice and its effect upon fertility. Immunized animals exhibited higher anti-proacrosin antibodies levels than controls (indirect ELISA), both in serum (p<0.01) and in seminal vesicle fluid (SVF; p<0.05). IgG2a levels were higher than IgG1 in serum (p<0.01) and similar in SVF. IL-10 and TGF-ß1 mRNA levels were lower in testis (p<0.05), whereas TNF-α and IFN-γ transcript levels were increased in SV tissue (p<0.05). Immunized mice showed a trend toward higher IFN-γ concentration in serum and SVF than controls. Male fertility rate was diminished in immunized mice (p<0.01) and inversely correlated with serum and SVF anti-proacrosin IgG levels (p<0.001). Immunized animals also had fewer pups born than controls (p<0.01). To our knowledge, this is the first report on DNA immunization done in CF1 mice. Injection of proacrosin DNA induces an immune response in the male reproductive tract characterized by high levels of specific antibodies and cytokine changes. These factors may alter the crucial balance of the genital tract microenvironment required for adequate fertilization and pregnancy.

Detection of Staphylococcus aureus adhesion and biofilm-producing genes and their expression during internalization in bovine mammary epithelial cells.

Staphylococcus aureus is one of the most prevalent pathogens isolated from bovine mastitis, causing chronic intramammary infections (IMI) that limit profitable dairying. The course of infection is often associated with factors both related to the host and the bacterium. Aims of this study were to select S. aureus isolates from bovine IMI with different genotypic profiles harboring genes involved in adherence and biofilm production, to determine the behavior of these strains in contact with bovine mammary epithelial cells (MAC-T) and the expression of those genes during bacterial-cell early interactions. The genetic diversity of 20 S. aureus strains that were isolated from milk samples taken from cows with persistent-P and non-persistent-NP IMI was high, discriminated into 13 fingerprint groups. The occurrence of genes coding for S. aureus surface proteins (clfA, clfB, fnbA, fnbB, fib, cna) and biofilm formation (icaA, icaD, icaC, bap) and in vitro biofilm-forming ability was not related to strain clinical origin (NP or P). Internalization of S. aureus into MAC-T cells was strain-dependent and internalized bacteria overexpressed adherence and biofilm-forming genes compared with those that remained in the supernatant of co-cultures; particularly those genes encoding FnBPs and IcaD. Strains yielding highest invasion percentages were those able to overexpress fnBP, irrespectively of the presence of other evaluated genes. Strains from NP IMI showed a greater multiplication capacity in vitro compared with strains from P IMI. These results provide new insights about S. aureus differential gene expression of adhesion-internalization factors during early interaction with mammary epithelial cells.