Incomplete Freund's Adjuvant Group Research Articles

Production, purification, and in vivo evaluation of a novel multiepitope peptide vaccine consisted of immunodominant epitopes of SYCP1 and ACRBP antigens as a prophylactic melanoma vaccine

Melanoma cells are significantly resistance to the current treatments. Therefore, the best option for high-risk populations is prevention. Recently, many preventive cancer vaccines have been developed. In our previous study, several bioinformatic tools were employed for selection of the most immunodominant epitopes of acrosin binding protein (ACRBP) and synaptonemal complex protein 1 (SYCP1) antigens to design multiepitope DNA and peptide cancer vaccines. In the current study, the final construct of the multiepitope DNA vaccine was placed into a pcDNA3.1 vector and then, subcloned into a pET-28a (+) expression vector for transfecting BL21 E. coli strain. The recombinant multiepitope peptide vaccine, weighing 6.35 kDa, was purified by Fast protein liquid chromatography technique (FPLC) and detected by western blotting. Subsequently, C57BL/6 mice were immunized by a mixture of the peptide vaccine and incomplete Freund's adjuvant (IFA) (four vaccinations with one-week intervals). Two weeks after the last vaccination, the serum levels of the peptide-specific IgG total, IgG2a, and IgG1 were measured by enzyme-linked immunosorbent assays (ELISA). Also, the immunized mice splenocytes efficacy for producing interleukin-4 (IL-4) and interferon-γ (IFN-γ) after stimulation with the peptide vaccine was evaluated. At last, the prophylactic effect of the peptide vaccine immunization was evaluated in B16-F10 murine melanoma model. The peptide vaccine immunization caused a significant increase in the serum levels of IgG1, IgG2a, and IgG2a. Also, the immunized mice splenocytes exhibited significantly higher ability to produce IL-4 (10-fold) and IFN-γ (16-fold) after stimulation with the peptide vaccine, in comparison with the PBS and IFA groups. The peptide immunized mice exhibited 50.2% and 43% decrease in the mean tumors' volume in comparison with PBS and IFA groups. Also, the mean survival time for the peptide immunized mice was 33 ± 1.3 days which was 5 and 6 days more than the PBS and IFA groups, respectively. The obtained results exhibit high efficacy of the designed multiepitope peptide vaccine for the immune system activation and anti-tumor prophylactic effects in the murine melanoma model.

IL-17A Can Promote Propionibacterium acnes-Induced Sarcoidosis-Like Granulomatosis in Mice.

The etiology of sarcoidosis is unknown. In this study, Propionibacterium acnes (PA) was used to induce sarcoidosis-like granulomatous inflammation in a mouse model. Wild-Type (WT) C57BL/6 mice were divided into three groups: (1) WT-PA group; (2) WT-PA + Incomplete Freund's Adjuvant (IFA) group; and (3) WT-PBS group. Loose granuloma formation was observed in the lungs on day 56 in the WT-PA and WT-PA + IFA groups. The proportions of peripheral Th17 cells in the WT-PA (p = 0.0004) and WT-PA + IFA groups (p = 0.0005) were significantly higher than that in the WT-PBS group. The proportions of peripheral Treg cells in the WT-PA (p < 0.0001) and WT-PA + IFA groups (p < 0.0001) were lower than that in the WT-PBS group. Then, to explore the mechanism of IL-17, Wild-Type (WT) C57BL/6 mice were divided into three groups: (1) WT-PBS group (2) WT-PA group; (3) WT-PA + mouse IL-17A neutralizing antibody (IL-17Ab) group. IL-17A gene knockout mice (KO) were divided into two groups: (1) KO -PA group; (2) KO-PBS group. The KO-PA and WT-PA + IL-17Ab groups showed reduced inflammation and no loose granuloma formation on day 56. As compared to the WT-PA group, the ratio of peripheral Th17 in the KO-PA (p < 0.0001) and WT-PA + IL-17Ab groups (p < 0.0001) decreased, while the ratio of peripheral Treg in the KO-PA (p < 0.0001) and WT-PA + IL-17Ab (p = 0.0069) groups increased on day 56. Hence, PA can be used to establish a mouse model of sarcoidosis-like granuloma. IL-17A plays an important role in experimental sarcoidosis-like granuloma formation.

Effects of adjuvants for human use in systemic lupus erythematosus (SLE)-prone (New Zealand black/New Zealand white) F1 mice

The safety of four different adjuvants was assessed in lupus-prone New Zealand black/New Zealand white (BW)F1 mice. Four groups of mice were injected intraperitoneally with incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), squalene (SQU) or aluminium hydroxide (ALU). An additional group received plain phosphate-buffered saline (PBS) (UNT group). Mice were primed at week 9 and boosted every other week up to week 15. Proteinuria became detectable at weeks 17 (IFA group), 24 (CFA group), 28 (SQU and ALU groups) and 32 (UNT group). Different mean values were obtained among the groups from weeks 17 to 21 [week 17: one-way analysis of variance (anova) P = 0·016; weeks 18 and 19: P = 0·048; weeks 20 and 21: P = 0·013] being higher in the IFA group than the others [Tukey's honestly significant difference (HSD) post-test P < 0·05]. No differences in anti-DNA antibody levels were observed among groups. Anti-RNP/Sm antibody developed at week 19 in only one CFA-treated mouse. Mean mouse weight at week 18 was lower in the ALU group than the IFA (Tukey's HSD post-test P = 0·04), CFA (P = 0·01) and SQU (P < 0·0001) groups, while the mean weight in the SQU group was higher than in the IFA (P = 0·009), CFA (P = 0·013) and UNT (P = 0·005) groups. The ALU group weight decreased by almost half between weeks 29 and 31, indicating some toxic effect of ALU in the late post-immunization period. Thus, SQU was the least toxic adjuvant as it did not (i) accelerate proteinuria onset compared to IFA; (ii) induce toxicity compared to ALU or (iii) elicit anti-RNP/Sm autoantibody, as occurred in the CFA group.

Effect of Rolipram, a Phosphodiesterase IV Inhibitor, on Allergic Footpad Swelling using Various Adjuvants in Mice

We studied the effect of rolipram, a phosphodiesterase (PDE) IV inhibitor, on allergic footpad swelling in mice. For this study, varying adjuvants including complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and Imject Alum (Alum) were used because the extent of antigen-specifically induced T helper type 1 (Th1) and Th2 responses had been shown to depend on adjuvants used. To induce allergic footpad swelling, we immunized mice with ovalbumin (OVA) emulsified in either CFA or IFA, dissolved in Alum or in phosphate-buffered saline (PBS) as a control (day 0), followed by subcutaneous injection of the antigen into footpads on day 21. Rolipram was given orally to the animals daily from days 0-20. Results showed that treatment with rolipram was followed by an increase in early swelling at 0.5 h and a decrease in late swelling at 6 and 24 h in the CFA group. In the IFA group, rolipram significantly enhanced swelling at, but not after, 30 min. In the Alum and the PBS groups, the PDE inhibitor failed to affect the OVA-specific footpad reaction at all times examined. Treatment of the CFA and IFA groups with rolipram significantly inhibited the production of the Th1 antibody anti-OVA immunoglobulin G2a (IgG2a), and the drug enhanced Th2 cell-dependent anti-OVA IgE production. In both groups, rolipram also enhanced the secretion of Th2 cytokines including interleukin-4 (IL-4) and IL-10. These findings suggest that rolipram may facilitate early allergic footpad swelling mediated by Th2 immune responses, while the late phase of swelling associated with Th1 responses may be attenuated by the PDE IV inhibitor.

DP020 Establishment of a Capture-ELISA to detect the Beta2-toxin of Clostridium perfringens

A selected water-in-oil (W/O) emulsion was evaluated in terms of stability and antibody-response and side-effects in experimental animals.The results from stability tests performed at 4°C and 37°C indicate a storability of the W/O emulsion of more than a year under normal storage conditions. The antibody-response of rabbits after injection of E.coli K99 antigen and bovine serum albumin (BSA) using the W/O emulsion as vehicle was generally significantly higher than the response to the same antigens injected within incomplete Freund's adjuvant (IFA): twenty days after the first antigen injection the rabbits injected with antigen in the W/O emulsion showed average anti-K99 and anti-BSA titres which were ca 5x as high as the titres in sera from rabbits injected with the antigens in IFA. Thirty-two and forty-two days after the first antigen injections the W/O group showed anti-K99 titres which were respectively ca 3.5x and 2x higher than titres in the IFA group, while anti-BSA titres in the W/O group were respectively ca 13x and 6.5x higher than in the IFA group.Intra-muscular and/or subcutaneous injection of the W/O emulsion in rabbits and pigs resulted in a slight degeneration of local tissue. When antigens were present in the emulsion granulomatas were often observed. The rise in temperature in pigs after injection of killed E.coli 0149:K91,K88 in the W/O emulsion was comparable with that induced by E.coli in physiological saline.From these results it is concluded that the W/O emulsion is likely to be a promising basis for veterinary vaccines and that the application of the W/O emulsion may contribute to the limitation of the number of animals used for raising antibodies for general immunological purposes.

Single injection of insulin delays the recurrence of diabetes in syngeneic islet-transplanted diabetic NOD mice.

Insulin has been implicated in the pathogenesis of type 1 diabetes and oral administration of insulin has been shown to delay the onset of diabetes in NOD mice. In this study we determined whether a single footpad injection of insulin will protect syngeneic islet grafts from autoimmune destruction when placed under the kidney capsule of diabetic NOD mice. Five hundred islets were transplanted under the kidney capsule of diabetic female NOD mice in conjunction with a single footpad injection of either pork insulin in saline or mixed with incomplete Freund's adjuvant (IFA). Control groups received either IFA or saline alone. Seven of 11 animals (63.6%) given insulin in IFA exhibit long-term graft survival (>75 days; mean +/- SEM >85.4+/-16.1) whereas only 3 of 12 animals (25.0%) in the IFA group had graft survival longer than 75 days (mean +/- SEM >41.9+/-12.8 days). In contrast, none of the animals that received insulin in saline (17.3+/-2.5 days) and saline only (16.1+2.0 days) exhibit prolonged graft survival. These results suggest that a single footpad injection of insulin can protect the islet graft from immune attack in NOD mice.

Immunization with Pseudomonas aeruginosa vaccines and adjuvant can modulate the type of inflammatory response subsequent to infection

Pseudomonas aeruginosa is the predominant pathogen in patients with cystic fibrosis (CF). To study the possibility of preventing lung inflammation and decreasing the progression of the infection by vaccination, we have developed a rat model of chronic P. aeruginosa lung infection. Rats were immunized with P. aeruginosa whole-cell sonicates, O-polysaccharide toxin A conjugate, an alginate-toxin A conjugate, or native alginate. Control animals received sterile saline or incomplete Freund's adjuvant (IFA). The macroscopic (mean score, 2.4 versus 2.7 to 3.2) (P < 0.05) and microscopic (mean score, 2.0 versus 2.1 to 2.8) pathologic abnormalities were less severe in the control rats injected with sterile saline than in the immunized rats and the IFA group. The more severe lung abnormalities observed in immunized rats could be due to the result of immune complex-mediated lung tissue damage. The histopathologic results in the saline control rats were characterized by acute inflammation dominated by numerous polymorphonuclear leukocytes surrounding the alginate beads (microcolonies), as in CF patients. In contrast, the inflammatory response in the IFA group and in the immunized rats had changed from an acute-type inflammation to a chronic-type inflammation dominated by mononuclear leukocytes and scattered granulomas. Cross-reacting antibodies were induced by the two alginate vaccines, and most immunized animals developed a significant (P < 0.001) antibody titer elevation (in enzyme-linked immunosorbent assay) of the immunoglobulin M (IgM), IgG, and IgA classes against the homologous antigens. The bacterial clearance was significantly (P < 0.05) more efficient in most immunized rats than in the control rats given sterile saline. The present study shows that none of the vaccines could completely prevent chronic lung inflammation 4 weeks after challenge. However, the changed pathologic condition in immunized rats to a chronic-type inflammation might be of great benefit in future management of CF patients since the developing lung tissue damage has been shown to be caused by polymorphonuclear leukocyte-released elastase.

High-titer HIV-1 neutralizing antibody response of rhesus macaques to gp160 and env peptides.

Three groups of four rhesus macaques were immunized twice, one month apart with purified recombinant HIV-1LAI gp160 in the presence of either alum, incomplete Freund's adjuvant (IFA), or SAF-1. Two months later, the animals were injected twice again with a synthetic peptide with the sequence of the principal neutralization determinant (PND) of the HIV-1LAI isolate mixed with the same adjuvants. All animals received a booster injection of gp160 and PND peptide at 6 months. This regimen of immunization induced in the SAF-1 and IFA groups a high-titer neutralizing antibody response that declined progressively over the course of the following 6 months. In contrast, only a weak response was observed in the alum group. Neutralizing antibody titers varied as anti-PND titers, suggesting that they were principally targeted to the PND. A shortened immunization protocol comprising two injections of gp160 at 0 and 1 month followed by one injection of PND peptide at 3 months is suggested as optimal for the induction of high titers of HIV-1 neutralizing antibodies in primates.

A selected water-in-oil emulsion: Composition and usefulness as an immunological adjuvant

A selected water-in-oil (W/O) emulsion was evaluated in terms of stability and antibody-response and side-effects in experimental animals. The results from stability tests performed at 4°C and 37°C indicate a storability of the W/O emulsion of more than a year under normal storage conditions. The antibody-response of rabbits after injection of E.coli K99 antigen and bovine serum albumin (BSA) using the W/O emulsion as vehicle was generally significantly higher than the response to the same antigens injected within incomplete Freund's adjuvant (IFA): twenty days after the first antigen injection the rabbits injected with antigen in the W/O emulsion showed average anti-K99 and anti-BSA titres which were ca 5x as high as the titres in sera from rabbits injected with the antigens in IFA. Thirty-two and forty-two days after the first antigen injections the W/O group showed anti-K99 titres which were respectively ca 3.5x and 2x higher than titres in the IFA group, while anti-BSA titres in the W/O group were respectively ca 13x and 6.5x higher than in the IFA group. Intra-muscular and/or subcutaneous injection of the W/O emulsion in rabbits and pigs resulted in a slight degeneration of local tissue. When antigens were present in the emulsion granulomatas were often observed. The rise in temperature in pigs after injection of killed E.coli 0149:K91,K88 in the W/O emulsion was comparable with that induced by E.coli in physiological saline. From these results it is concluded that the W/O emulsion is likely to be a promising basis for veterinary vaccines and that the application of the W/O emulsion may contribute to the limitation of the number of animals used for raising antibodies for general immunological purposes.