PML-RARA-targeted DNA vaccine induces protective immunity in a mouse model of leukemia

Despite improved molecular characterization of malignancies and development of targeted therapies, acute leukemia is not curable and few patients survive more than 10 years after diagnosis. Recently, combinations of different therapeutic strategies (based on mechanisms of apoptosis, differentiation and cytotoxicity) have significantly increased survival. To further improve outcome, we studied the potential efficacy of boosting the patient's immune response using specific immunotherapy. In an animal model of acute promyelocytic leukemia, we developed a DNA-based vaccine by fusing the human promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) oncogene to tetanus fragment C (FrC) sequences. We show for the first time that a DNA vaccine specifically targeted to an oncoprotein can have a pronounced effect on survival, both alone and when combined with all-trans retinoic acid (ATRA). The survival advantage is concomitant with time-dependent antibody production and an increase in interferon-gamma (IFN-gamma). We also show that ATRA therapy on its own triggers an immune response in this model. When DNA vaccination and conventional ATRA therapy are combined, they induce protective immune responses against leukemia progression in mice and may provide a new approach to improve clinical outcome in human leukemia.     

Schistosoma japonicum: a method for transformation by electroporation

Despite of our knowledge of genetic make up of schistosomes, a number of genes have not been characterized largely due to lack of effective transformation protocols. Here we present electroporation as a strategy for effective introduction of plasmids DNA into schistosomula and adults. Using plasmids of pEGFP-C1 as an expression vector, we first verified that the CMV promoter could direct EGFP to express in primary culture cells from Schistosoma japonicum. Subsequently, the plasmids were introduced into schistosomula and adults by electroporation and EGFP expression was demonstrated using molecular and microscopical methods. Our findings indicate that electroporation is an effective method for transformation of S. japonicum.     

Schistosoma mansoni-New Zealand rabbit model: resistance induced by infection followed by active immunization with protective antigens

In previous studies, rabbits immunized with adult worm antigens released from fresh adult schistosomes incubated in saline media showed a significant level of protection against challenge parasites. Focusing on the rabbit-Schistosoma mansoni model, concomitant immunity was investigated. A peculiar form of response to cercarial infection was observed: rabbits subjected to percutaneous infection and similar reinfections at different times after primary infection killed schistosomula from the challenge infection as well as established parasites from the primary infection. In this study the challenge infection stimulus was replaced by active immunization with an adult worm-derived protective antigenic mixture. The results show that immunization of New Zealand rabbits with an adult worm antigenic extract is capable of inducing a response that results in a significant reduction of the mean worm burden of the primary infection earlier than did homologous infection, as compared to worm reduction due to a second infection.     

Mechanisms of protective immunity in Hymenolepis nana/mouse model.

Some immunological and parasitological aspects related to the infection of Hymenolepsis nana in mice are summarized in this review, focusing on the immune effector mechanisms involved in this host/parasite relationship. H. nana is a small cestode tapeworm of man and mice. A primary egg-infection determines within few days a strong immunity. Immunity elicited by low-level primary infection is effective as a high-level infection. The protective role of both humoral and cell-mediated immunity is summarized. The histological findings demonstrate that eosinophils and mast-cells are implicated as effector cells. This review is an attempt to re-examine, at low-level infection, the immune mechanisms in H. nana/mouse model.     

Suppression of adaptive immunity to heterologous antigens by SJ16 of Schistosoma japonicum

Despite the great effort that has been given to control the disease, schistosomiasis remains the most important human helminth infection in terms of morbidity and mortality. Natural infection of schistosomes induces very little protective immunity against reinfection. Moreover, effective schistosome vaccines for practical use have not been developed. These parasites appear to have evolved highly effective modulatory mechanisms on their host's immune system that promote the parasites' survival and also hinder the development of effective strategies for treatment of the disease. Understanding of the mechanisms of schistosome-mediated immune modulation would be most helpful in schistosomiasis prevention and control. Previously, we have identified from Schistosoma japonicum an anti-inflammatory protein, Sj16, which suppresses thioglycollate-induced peritoneal inflammation in BALB/c mice, as well as thioglycollate-mediated peritoneal macrophage maturation, while modulating cytokine and chemokine production from peritoneal cells. In the present study, we have further investigated the modulatory effect of Sj16 on the host's adaptive immunity to heterologous antigens with the use of recombinant Sj16 (rSj16) expressed and purified from Escherichia coli . Results from this study indicate that rSj16 significantly suppresses antibody production, in addition to Th1 and Th2 responses to heterologous antigens in the BALB/c mouse model. Our study also reveals that rSj16 suppresses lipopolysaccharide-induced major histocompatibility complex II expression and IL-12 production, while increasing IL-10 production in resident peritoneal macrophages. These results may partially explain why parasite-related antigens cannot mount a protective immunity during early stages of schistosome infection.     

The miniature pig: a unique experimental model for Schistosoma japonicum infection

As part of a search for good animal models for human schistosomiasis, two miniature pigs of the CLAWN strain (C-1, C-2) were inoculated percutaneously with 200 Schistosoma japonicum cercariae of the Chinese strain, and the subsequent infection was monitored parasitologically, pathologically and serologically. Egg excretion into feces began at 5 weeks post-infection (p.i.) and became pronounced from 8 weeks to 17–20 weeks p.i. The average number of eggs in 1 g feces of each pig at the peak period between 8 and 20 weeks were 288 and 277, respectively. C-1 and C-2 were killed and perfused at 27 and 47 weeks p.i. and adult worm numbers recovered were 35 and 15, respectively. C-2 had at least four pairs of viable mature worms but no detectable fecal eggs for a month before perfusion, suggesting that any produced eggs were not excreted into the feces during this period. Egg deposits associated with inflammatory reactions were observed by histological examination of the liver, spleen, pancreas, mesenteric lymph nodes, lung, and small intestine. This suggests that reduced fecal excretion of eggs into the feces did not correlate to reduced parasite numbers in the chronic phase of schistosomiasis. This is the first report showing the miniature pig to be a potential model for human S. japonicum infection.     

Characteristics of protective immunity in mice induced by drug-attenuated larvae of Schistosoma mansoni. Antigen localization and antibody responses

Partial resistance to reinfection developed in mice with immature infections of Schistosoma mansoni treated with the drug Ro11-3128, whereas mice vaccinated with irradiated cercariae and treated in a similar way did not become immune. Persistence of drug-treated parasites in the skin and draining lymph nodes, which prolonged the opportunity for efficient Ag presentation, was necessary for the development of protective immunity. Death and clearance of parasites solely in the skin, was not sufficient to induce protection. The expansion in the number of lymphocytes in the skin-draining nodes after vaccination, was reflected in the contrasting levels of resistance induced by the different drug-treatment regimes. Challenge parasites were eliminated predominantly after they reached the lungs. An investigation of antibody reactivity revealed an immunodominant response against a doublet of Ag of Mr 97 to 99 kDa. Recognition of this complex by antisera from different groups of mice was not related to their immune status. Western blots and inhibition analysis showed that this doublet has epitopes in common with the Sm97/paramyosin protective Ag, originally identified by antisera reactivity from mice immunized with a nonliving vaccine.     

Cloning, expression and protective immunity evaluation of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum

1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3′ and 5′ ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2 pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokary- otic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coli. SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (posi- tive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also sug- gested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity.     

Cloning, expression and protective immunity evaluation of the full-length cDNA encoding succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum

1071-bp fragment was obtained from the Schistosoma japonicum (Chinese strain) adult cDNA library after the 3' and 5' ends of the incomplete expression sequence tag (EST) of succinate dehydrogenase iron-sulfur protein of Schistosoma japonicum (SjSDISP) were amplified by the anchored PCR with 2pairs of primers designed according to the EST of SjSDISP and the sequence of multiclone sites of the library vector. Sequence analysis indicated that the fragment was a full-length cDNA with a complete open reading frame (ORF), encoding 278 amino acid residues. The fragment was cloned into prokaryotic expression vector pQE30, and subsequently sequenced and expressed in Escherichia coll.SDS-PAGE and Western-blot analyses showed that the recombinant protein was about 32 kD and could be recognized by the polyclonal antisera from rabbits immunized with Schistosoma japonicum adult worm antigen. Compared with the FCA controls, mice vaccinated with rSjSDISP (test) or rSjGST (positive control) all revealed high levels of specific antibody and significant reduction in worm burden, liver eggs per gram (LEPG), fecal eggs per gram (FEPG) and intrauterine eggs. These results suggest that SjSDISP may be a novel and partially protective vaccine candidate against schistosomiasis. In contrast to the worm burden reduction rate, the higher degree of egg reduction rate in the test group also suggested that SjSDISP vaccine may primarily play a role in anti-embryonation or anti-fecundity immunity.     

Production and properties of a mouse monoclonal IgE antibody to Schistosoma japonicum

A monoclonal IgE antibody was prepared by fusion of NS-1 myeloma cells with spleen cells of C3H/He mice immunized with an extract of adult worms of Schistosoma japonicum (Sj). The antibody was able to elicit passive cutaneous anaphylaxis in the rat skin against Sj with the highest titer of 1/256,000 in an ascitic form but did not cross-react with any of antigens extracted from S. mansoni, Fasciola hepatica, Paragoniumus westermani, or Trichinella spiralis. Western blot analysis indicated that the monoclonal IgE antibody recognized epitopes on molecules of 82 kDa, 97 kDa, 160 kDa, and 200 kDa, at least some of which were recognized by IgG antibodies of patients with chronic schistosomiasis japonica. The IgE antibody also recognized a 97-kDa antigen expressed on the surface of mechanically transformed schistosomula. Passive transfer of the antibody into mice in an early stage of challenge infection resulted in a partial but significant reduction of recovery of adult worms. However, similar treatment was not effective for the protection if the antibody was given in the postlung stage of the infection. Moreover, eosinophil-mediated damage to schistosomula was observed in vitro in the presence of the monoclonal anti-Sj IgE antibody, whereas the damage was not observed in the presence of another monoclonal IgE antibody with dinitrophenyl specificity.     

Plasmodium berghei: antibody response and protective immunity induced by vaccination

The authors have studied the behaviour of Swiss mice and of 5 inbred strains of mice in order to investigate: the protective effect, in the homologous infection test, of six vaccine inoculations of irradiated parasites belonging to two strains of Plasmodium berghei: ISTISAN and K173; the capacity to produce humoral antibodies after vaccine treatments and during infection; the probable correlation between the high antibody titre and the protection against infection. The results of the present study show that the antibody response plays a precise role in the immunity induced by vaccination. There is a certain degree of correlation, which is more evident for K173 vaccine, between the level of antibody response during infection and the protective efficacy of vaccination.     

T cell epitope-based peptide-DNA dual vaccine induces protective immunity against Schistosoma japonicum infection in C57BL/6J mice

Schistosomiasis is a major public health problem that primarily affects developing countries. Although schistosomicidal drugs exist, the development of an efficacious vaccine would potentially be the most powerful means of controlling this disease. Previous studies have shown that vaccination with selected protective epitopes successfully induced partial protection and/or reduced female fecundity in animal models. Thus, we investigated whether the T cell epitope P5 from the host-interactive tegument of Schistosoma japonicum 22.6 (S. japonicum) could act as a protective epitope. The protective potential of P5 in a vaccine against S. japonicum was determined by using a T cell epitope based peptide-DNA dual vaccine (PDDV). In our experiments, the vaccine construct (P5-18K-PDDV) contains the peptide of the T cell epitope (P5) and plasmid DNA, encoding P5 and adjuvant GM-CSF. We show that P5-18K-PDDV induced both cell-mediated and humoral immune responses in vivo and achieved partial protection against S. japonicum infection in C57BL/6J mice. Histopathological studies reveal that P5-18K-PDDV immunized mice had substantially reduced liver pathology compared to the control groups. Together, these results suggest that P5 could be used as a vaccine immunogen for both worm killing and disease prevention against S. japonicum.     

Induction of protective immunity against Schistosoma mansoni by a nonliving vaccine. II. Response of mouse strains with selective immune defects

The efficacy of a new vaccination procedure against Schistosoma mansoni, involving intradermal injection of nonliving antigen combined with the bacterial adjuvant Mycobacterium bovis strain bacillus Calmette Guérin, was tested in several strains of mice. Development of protection against subsequent infection was compared with in vivo skin test reactivity and in vitro humoral reactivity to soluble and surface-associated schistosome antigens. Significant levels of resistance and immune response were displayed by many inbred mouse strains, including C57BL/6J, C3H/HeN, and CBA/J, as well as outbred Swiss-Webster mice. However, no definite correlation was observed between the level of any particular immune response and the level of resistance to challenge S. mansoni infection. Development of protective immunity was also examined in mice with various immune defects, to determine whether these responses are relevant to resistance in this model. Animals with defective specific immediate hypersensitivity response due to deficiencies in IgE (SJL/J) or mast cell (W/Wv) production displayed strong resistance as a result of immunization. Likewise, mice bearing the lpsd (C3H/HeJ) or xid (CBA/N) mutations, affecting cellular or humoral response to certain thymus-independent antigens, developed significant levels of resistance after immunization. A/J mice, with defects in cellular recognition of bacterial endotoxin as well as deficiencies in natural killer cell activity and complement function, also showed significant protective immunity. Thus, these reactivities do not appear to be essential to the resistance against S. mansoni induced by the nonliving vaccine. Two nonresponder strains were identified, P and BALB/c. P mice were defective in specific delayed hypersensitivity response as well as resistance to infection. However, BALB/c mice showed no obvious immune deficiencies at the time of challenge. These results agreed with previous findings in mice immunized by exposure to radiation-attenuated cercariae with one exception; BALB/c mice were protected by vaccination with irradiated cercariae but not by the nonliving vaccine. Thus, further examination of immune response in mice identified in this study as high and low responder strains should allow characterization of critical immune resistance mechanisms induced by the nonliving vaccine, as well as immune mechanisms operating in common between these two models of resistance to S. mansoni.     

Host identification by Schistosoma japonicum cercariae

Attachment, the first phase of host identification by Schistosoma japonicum cercariae, can occur in 2 different ways. Cercariae clinging to the water surface simply swing around and transfer to the host skin. Free-swimming cercariae behave like S. mansoni: upon touching a substrate, they switch from tailward to forward movement, swim in an arc, and attach to it with the penetration organ. Neither type of attachment is influenced by chemical, thermal, or specific mechanical stimuli from the host. The second phase, remaining on the host, requires a solid hydrophobic surface and seems to depend only on the cercaria's ability to cling to it. This phase is not influenced by chemical or thermal stimuli. The third phase, creeping across the host surface, is independent of chemical and mechanical stimuli. Cercariae migrate in thermal gradients to a preferred temperature of 37 +/- 3 C and then attempt to penetrate. Penetration, the fourth phase, was evoked by human skin surface lipids. The free fatty acid (FA) fraction was identified as exclusively stimulating components. Saturated FA's were effective at chain lengths between 10 and 14 carbon atoms (pH 7.0), and unsaturated FA's were effective at longer chains and their activity increased with increasing number of double bonds. Dog skin surface components did not stimulate cercarial penetration, which can be attributed to the lack of free FA's. A temperature of 32-40 C also stimulated penetration responses, which might be the main stimulus in animal hosts, whose skin surfaces contain no or only a few free FA's. FA's and heat evoked a transformation of cercarial tegument simultaneous with penetration behavior, making the organisms osmotically sensitive. The host identification of S. japonicum cercariae is very nonspecific compared with the differentiated host recognition of S. mansoni.