Vaccination of mice with a 30 kDa Schistosoma antigen with and without human adjuvant induces high protection against S. mansoni infection

A 30 kDa antigen was characterized as a hydrophobic polypeptide containing 16 amino acids and evaluated as a potential candidate vaccine against infection by Schistosoma mansoni. CD1 albino mice immunized at 0, 14, and 21 days with 25 or 50 microg of the 30 kDa antigen per mouse with and without alum developed high levels of IgG antibodies (predominantly IgG2a and IgG2b isotypes). When immunized mice were infected with 200 S. mansoni cercariae, the highest protection levels (61% and 65% reduction in worm burden in two separate experiments) were obtained using the 50-microg antigen without alum adjuvant. The granuloma size decreased to 10%, a non-significant level in mice immunized using alum adjuvant. The results demonstrate the ability of the 30 kDa antigen with and without alum adjuvant to protect mice against S. mansoni infection.     

Live and killed vaccines against toxoplasmosis in mice

Mice were immunized with live organisms of the different stages (i.e., tachyzoites, bradyzoites, or sporozoites) of Toxoplasma gondii, or with killed tachyzoites with or without adjuvants. The adjuvants used were liposomes, anhydrides of myristic or lauric acid, levamisole and Freund's complete or incomplete adjuvant. The following strains of T. gondii were used: RH, M-7741, the nonpersisting, temperature-sensitive mutants ts-1, ts-4, or ts-5, and the "back mutant" of ts-1 (Pfefferkorn and Pfefferkorn, 1976). The protection afforded was measured by challenge with the pathogenic M-7741 strain. Killed tachyzoites alone, or with adjuvants, offered only slight protection against challenge with M-7741 and no protection against challenge doses that were lethal to all control mice. Chronic infection and live nonpersisting vaccines conveyed a strong immunity to challenge, except strain ts-1. Because it was less pathogenic and did not require chemoprophylaxis, strain ts-4 best fulfilled the requirements for a good vaccine; its effect in hosts other than the mouse remains to be determined. The immunity induced by tachyzoites, bradyzoites, or sporozoites appeared equally strong when challenged with sporozoites.     

Pathology associated with vaccination against Schistosoma mansoni in mice using cryopreserved radiation-attenuated schistosomula

Twenty-one mice were injected intramuscularly with 2000 Schistosoma mansoni schistosomula irradiated at 20 krad and cryopreserved; three mice were killed on each of days 0, 2, 5, 9, 19, 28 and 44 days after infection and muscle from the site of injection in the left hind leg, the lungs and livers removed for histological examination. Schistosomula were seen in sections from the leg muscle from days 0 to 19 inclusive, in the lungs from day 2 to day 28 inclusive and in the livers from days 9 to 28 inclusive. Most schistosomula were seen in sections of the leg muscle with considerably fewer parasites occurring in the lungs and especially the livers. Granulomatous reactions comprising eosinophils, polymorphs, plasma cells and macrophages were first seen in the leg muscle on day 2, in the lungs on day 5 and in the liver on day 19. The peak inflammatory reactions appeared to occur between days 5 and 9, 9 and 19 and 28 and 44 respectively in the three tissues. The pathology is discussed in relation to the dose of irradiation required to attenuate the schistosomula for optimal immunogenicity.     

Vaccination in murine schistosomiasis with adult worm-derived antigens: variables influencing protection in outbred mice

Previous studies have shown that both permissive (mouse) and partially permissive (rabbit) hosts develop high levels of resistance against Schistosoma mansoni infection after vaccination with a multiple antigen extract (SE) obtained by incubation of living adult worms in saline, plus bacterial adjuvant. To investigate variables influencing SE-induced protection in murine schistosomiasis, a series of distinct vaccination protocols were performed focussing on the immunization dose, carrier systems, route, site and amplitude of challenge infection, and time between immunization and challenge. In addition, a new approach was adopted to evaluate SE protective activity, by means of population analysis of worm burden frequency distributions in a large scale study of vaccination in outbred Swiss mice. Distinct curves of frequency and a drastic difference in worm burden distribution of frequencies from SE-vaccinated x non-vaccinated mice were found. It was shown that SE could generate 75% mean protection in outbred mice even in the absence of adjuvant. In addition SE immunization was also able to induce full protection against lethal infection. SE-induced protection could be modulated by such parameters as dose of SE immunization/challenge interval, and route of cercariae injection. These data show that SE yields very high protective activity in outbred mice, and may provide a further insight for rational design of a vaccine in experimental schistosomiasis.     

Host resistance against Candida alibcans infection in mice with adjuvant induced arthritis

In mice and humans two distinct CD4+ helper T cells, known as Th1 and Th2 are identified. They are characterized by the different cytokine milleau they induce. The balance between Th1 and Th2 responses is thought to be decisive for the initiation and course of some autoimmune disorders, as well as for the outcome of infectious processes. In the present study the development of Candida alibcans infection in mice with adjuvant-induced arthritis was investigated. An impaired host resistance against C. alibcans in arthritic mice was registered when the inoculation was done during the early and the established phases of arthritis. In contrast, the slight elevation of the number of survivors was detected when the infection was induced at the peak of inflammation. These data correlated with the changes of serum TNF-α level and delayed type hypersensitivity (DTH) toC. alibcans. This revised version was published online in June 2006 with corrections to the Cover Date.     

Vaccination against lymphocytic choriomeningitis virus infection in MHC class II-deficient mice

The impact of prophylactic vaccination against acute and chronic infection in a Th-deficient host has not been adequately addressed because of difficulties in generating protective immunity in the absence of CD4(+) T cell help. In this study, we demonstrated that a broad CD8(+) T cell immune response could be elicited in MHC class II-deficient mice by vaccination with adenovirus encoding lymphocytic choriomeningitis virus (LCMV) glycoprotein tethered to MHC class II-associated invariant chain. Moreover, the response induced conferred significant cytolytic CD8(+) T cell-mediated protection against challenge with a high dose of the invasive clone 13 strain of LCMV. In contrast, vaccination with adenovirus encoding unlinked LCMV glycoprotein induced weak virus control in the absence of CD4(+) T cells, and mice may die of increased immunopathology associated with incomplete protection. Acute mortality was not observed in any vaccinated mice following infection with the less-invasive Traub strain. However, LCMV Traub infection caused accelerated late mortality in unvaccinated MHC class II-deficient mice; in this case, we observed a strong trend toward delayed mortality in vaccinated mice, irrespective of the nature of the vaccine. These results indicated that optimized vaccination may lead to efficient protection against acute viral infection, even in Th-deficient individuals, but that the duration of such immunity is limited. Nevertheless, for select immunodeficiencies in which CD4(+) T cell deficiency is incomplete or transient, these results are very encouraging.     

Vaccination with gamma-irradiated Neospora caninum tachyzoites protects mice against acute challenge with N. caninum

Neospora caninum, an apicomplexan parasite, is a leading cause of bovine abortions worldwide. The efficacy of gamma-irradiated N. caninum strain NC-1 tachyzoites as a vaccine for neosporosis was assessed in C57BL6 mice. A dose of 528 Gy of gamma irradiation was sufficient to arrest replication but not host cell penetration by tachyzoites. Female C57BL6 mice were vaccinated with two intraperitoneal inoculations of 1 x 10(6) irradiated tachyzoites at 4-wk intervals. When stimulated with N. caninum tachyzoite lysates, splenocytes of vaccinated mice, cultured 5 and 10 wk after vaccination, secreted significant (P<0.05) levels of interferon gamma, interleukin (IL)-10, and small amounts of IL-4. Antibody isotype-specific ELISA of sera from vaccinated mice exhibited both IgG1 and IgG2a isotypes of antibodies. Vaccinated mice were challenged intraperitoneally with 2 x 10(7)N. caninum tachyzoites. All vaccinated mice remained healthy and showed no obvious signs of neosporosis up to the 25th day post-challenge when the study was terminated. All unvaccinated control mice died within 1 wk of infection. Gamma-irradiated N. caninum tachyzoites can serve as an effective, attenuated vaccine for N. caninum.     

Prostaglandin modulation of airway inflammation and hyperresponsiveness in mice sensitized without adjuvant

As adjuvant during sensitization may cause unspecific immune reactions, the aim of the present study was to define the role of cyclooxygenase (COX) activity on airway inflammation and airway hyperresponsiveness (AHR) in an adjuvant-free allergic mouse model.Administration of diclofenac and indomethacin (non-selective COX inhibitors), FR122047 (COX-1 inhibitor) and lumiracoxib (selective COX-2 inhibitor) enhanced AHR. Only diclofenac and lumiracoxib reduced the inflammatory cell content of bronchoalveolar lavage (BAL). Moreover, levels of prostaglandins in BAL were reduced by indomethacin and FR122047 but were unaffected by lumiracoxib. However, compared with antigen controls, none of the COX inhibitors displayed major effects on the production of cytokines, smooth muscle mass, number of goblet cells and eosinophils, or collagen deposition in the airways.These data in mice sensitized without adjuvant support the fact that COX products have a general bronchoprotective role in allergic airway inflammation. Furthermore, the data suggest that COX-1 activity predominantly generates prostanoids in BAL, whereas COX-2 activity is associated with the accumulation of inflammatory cells in BAL. This study further supports that AHR on the one hand, and the inflammatory response and generation of prostanoids on the other, are dissociated and, at least in part, uncoupled events.     

Vaccination with major outer membrane protein proteosomes elicits protection in mice against a Chlamydia respiratory challenge

Vaccines formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) have so far been shown to elicit the most robust protection against this pathogen. nMOMP is a membrane protein and therefore, detergents are used to keep it in solution. Detergents however, have toxic effects. To address this limitation, we tested a nMOMP proteosome vaccine and compared its ability to elicit protection against nMOMP solubilized in the detergent Z3-14. The two preparations were formulated with or without CpG + Montanide (C/M). As a control antigen we used ovalbumin. Mice vaccinated with nMOMP developed strong humoral and cell mediated Chlamydia-specific immune responses. Based on the IgG2a/IgG1 levels in serum and amounts of IFN-γ in splenocytes supernatants the immune responses were predominantly Th1-biased. The animals were subsequently challenged intranasally with 2 × 10³ Chlamydia inclusion forming units (IFU) and the course of the infection was followed for 10 days when the mice were euthanized. Based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, mice immunized with nMOMP-Ps and nMOMP + Z3-14 adjuvanted with C/M showed the most robust protection. In summary, nMOMP-Ps should be considered as Chlamydia vaccine candidates.     

The influence of adjuvant on induction of protective immunity by a non-living vaccine against schistosomiasis

Mice were protected against subsequent infection with Schistosoma mansoni by intradermal or s.c. vaccination with killed schistosomula or soluble parasite extracts and bacillus Calmette-Guérin (BCG). Treatment with i.p. immunization was somewhat less effective, whereas i.m. vaccination failed to elicit protective immunity. The level of resistance induced by intradermal immunization was influenced by the strain of BCG used, and isolated BCG cell walls did not reliably substitute for whole BCG organisms as adjuvant. Bordetella pertussis vaccine and saponin were also able to function as adjuvants for protective immunity in this model, whereas other immunopotentiators including Corynebacterium parvum and aluminum hydroxide were ineffective. No correlation between resistance to challenge infection and antibody levels was detected. Animals immunized intradermally using either protective or non-protective adjuvants all showed minimal humoral reactivity against schistosomulum surface Ag but strong IgG response to soluble parasite components including paramyosin, which is the major serologically recognized Ag in mice vaccinated intradermally with schistosome Ag plus BCG and is protective in this model. In contrast, a strong correlation was observed between resistance and Ag-specific cell-mediated reactivity, including IFN production by T lymphocytes in vitro and macrophage activation in vivo. These results further substantiate the hypothesis that protection in this model is based on cell-mediated immune effector mechanisms. Moreover, they may be of general relevance in the design of vaccination protocols using other Ag or against other infectious agents.     

Immunization of mice with gamma-irradiated intramuscularly injected schistosomula of Schistosoma mansoni

The parameters involved in the induction of resistance against Schistosoma mansoni by injection of irradiated, artificially transformed schistosomula were studied in mice. Single intramuscular injections of 500 schistosomula exposed to radiation doses in the range 2.3 to 160 krad. resulted in significant protection (in the range 20 to 50% as assessed by reduced worm burdens) against a challenge infection administered at intervals from 3 to 24 weeks post-vaccination. However, schistosomula irradiated with 20 krad. consistently resulted in better protection than those exposed to either higher or lower radiation doses despite the persistence of stunted adults from the infections irradiated with 2.3 krad. Vaccination with 40 krad. schistosomula resulted in significant protection in terms of reduced worm and tissue egg burdens and increased survival following lethal challenge. Varying the number of irradiated schistosomula, the frequency and route of their administration, the site of challenge and the strain of host all failed to enhance the level of resistance. However, percutaneously applied, irradiated cercariae were found to be more effective in stimulating resistance (60%) than intramuscularly injected, irradiated schistosomula (40%).     

Pharmacokinetic aspects of tert-butylaminoethyl disulfide, an experimental drug against schistosomiasis in mice

A preliminary study of the pharmacokinetic parameters of t-Butylaminoethyl disulfide was performed after administration of two different single doses (35 and 300 mg/kg) of either the cold or labelled drug. Plasma or blood samples were treated with dithiothreitol, perchloric acid, and, after filtration, submitted to further purification with anionic resin. In the final step, the drug was retained on a cationic resin column, eluted with NaCl 1M and detected according to the method of Ellman (1958). Alternatively, radioactive drug was detected by liquid scintillation counting. The results corresponding to the smaller dose of total drug suggested a pharmacokinetic behavior related to a one open compartment model with the following parameters: area under the intravenous curve (AUCi.v.): 671 +/- 14; AUCoral: 150 +/- 40 micrograms.min.ml-1; elimination rate constant: 0.071 min-1; biological half life: 9.8 min; distribution volume: 0.74 ml/g. For the higher dose, the results seemed to obey a more complex undetermined model. Combining the results, the occurrence of a dose-dependent pharmacokinetic behavior is suggested, the drug being rapidly absorbed and rapidly eliminated; the elimination process being related mainly to metabolization. The drug seems to be more toxic when administered I.V. because by this route it escapes first pass metabolism, while being quickly distributed to tissues. The maximum tolerated blood level seems to be around 16 micrograms/ml.     

Immunization of mice with cells from juvenile worms of Schistosoma japonicum provides immunoprotection against schistosomiasis

To validate the protective efficacy against schistosomiasis by immunization with cells from juvenile Schistosoma japonicum in a murine model and to analyze possible factors related to protection, in this study, two independent repeated vaccination trials were performed. After three subcutaneous vaccina- tions, in trial one, in the absence of adjuvant, primary juvenile worm cells (pJCs) from S. japonicum induced remarkable average reductions in worm burden (54.3%), liver eggs per gram (LEPG) load (59.8%) as well as egg granulomas size (66.5%) compared to PBS control group (P<0.01), which were significantly higher than those elicited by fractions of juvenile worm cells (JCFs) or fractions of juvenile worms (JWFs) (P<0.05). Non-cell components of worms (WNCs) showed no significant protection. In trial two, compared to PBS control group, significant protective effect was also observed for cultured juvenile worm cells (cJCs) from S. japonicum with 58.4% worm reduction and 68.1% LEPG reduction (P<0.01). However, cultured adult worms cells (cACs) showed significantly higher worm burden (P<0.05) and egg burden (P<0.01) when compared to cJCs. Immunological analysis of trial two revealed that cJCs engendered a Th1-biased mixed Th1/Th2 type of immune response while cACs elicited a Th2-type response. Our data indicated that immunization with both primary and cultured cells from S. japonicum juvenile worms provided high immunoprotection, for which the physical character of immunogens, stage-specific parasite and the type of immune response induced might be responsible, suggesting that vaccination with whole cells from S. japonicum larvae is a promising approach to produce protec- tive immunity against schistosomiasis.     

Vaccination of mice against invasive aspergillosis with recombinant Aspergillus proteins and CpG oligodeoxynucleotides as adjuvants

In a murine model of invasive pulmonary aspergillosis, dendritic cells (DCs) pulsed with Aspergillus antigens induced the activation of CD4(+) Th1 cells capable of conferring resistance to the infection. Here we show that the combined, local delivery of unmethylated CpG oligodeoxynucleotides (ODNs) and the Asp f 16 Aspergillus allergen resulted in the functional maturation and activation of airway DCs capable of inducing Th1 priming and resistance to the fungus. Therefore, ODNs act as a potent adjuvant for the vaccine-induced protection against the fungus by promoting dominant Th1 response to Aspergillus antigens and allergens.     

Vaccination with epimastigotes of different strains of Trypanosoma rangeli protects mice against Trypanosoma cruzi infection

In our laboratory, we have developed a model of vaccination in mice with Trypanosoma rangeli, a non-pathogenic parasite that shares many antigens with Trypanosoma cruzi. The vaccinated mice were protected against infection with virulent T. cruzi. The goal of the present work was to study the protective activity of strains of T. rangeli of different origin, with the aim of analysing whether this protective capacity is a common feature of T. rangeli. BALB/c mice were vaccinated with live or fixed epimastigotes of two T. rangeli strains, Choachi and SC-58. Vaccinated (VM) and control mice (CM) were infected with virulent T. cruzi, Tulahuen strain. The results showed that the levels of parasitemia of VM, vaccinated with the two strains of T. rangeli were significantly lower than those developed in CM. The survival rate of VM was higher than that CM. Histological studies revealed many amastigote nests and severe inflammatory infiltrates in the heart and skeletal muscles of CM, whereas in the VM only moderate lymphomonocytic infiltrates were detected. Altogether, the results of the present work as well as previous studies show that the antigens involved in the protection induced by T. rangeli are expressed in different strains of this parasite. These findings could prove useful in vaccine preparation.     

Vaccination with cytoplasmic ErbB-2 DNA protects mice from mammary tumor growth without anti-ErbB-2 antibody.

Wild-type ErbB-2 (E2) positive D2F2/E2 tumors are rejected by active vaccination with ErbB-2 DNA. However, anti-ErbB-2 Ab response can cause cardiac toxicity or interfere with cellular immunity. It will be advantageous to induce only cellular immunity by active vaccination. A panel of E2 DNA vaccines were constructed, and their vaccination efficacy was ranked as E2 > tyrosine kinase-deficient ErbB-2 (E2A) > full-length ErbB-2 targeted to the cytoplasm (cytE2) > tyrosine kinase-deficient cytE2 (cytE2A). E2A is a tyrosine kinase-deficient mutant containing a single residue substitution. CytE2 or cytE2A encodes a full-length protein that is targeted to and rapidly degraded in the cytosol by the proteasomes. Covaccination with cytE2A and GM-CSF or IL-2 DNA resulted in equivalent anti-tumor activity as E2. However, anti-ErbB-2 Ab was induced by E2 or E2A, but not cytE2 or cytE2A. Therefore, cytE2A appears to induce anti-tumor immunity without an Ab response. ErbB-2-specific CTL were detected in mice immunized with cytE2A and GM-CSF and have rejected tumor challenge. Depletion of CD8, but not CD4 T cells reduced anti-tumor immunity, indicating CTL as the effector cells. Covaccination with E2A and cytE2A induced synergistic anti-tumor activity, supporting enhanced peptide presentation from cytE2A, which was further evidenced by superior CTL activation using APCs expressing cytE2 vs E2. Taken together, cytoplasmic ErbB-2 DNA induced anti-tumor CTL, but not humoral response, demonstrating the feasibility of eliciting individual effector mechanism by targeted DNA vaccine.