Vaccination with Leishmania soluble antigen and immunostimulatory oligodeoxynucleotides induces specific immunity and protection against Leishmania donovani infection

In this report, we investigated the effect of ODN containing immunostimulatory CG motifs as adjuvant with soluble antigen (SA) from Leishmania donovani. BALB/c mice were vaccinated with the soluble antigen with or without CpG-ODN as adjuvant and then challenged with L. donovani metacyclic promastigotes. CpG-ODN alone resulted in partial protection against challenge with L. donovani. Immunization of mice with SA and CpG-ODN showed enhanced reduction in parasite load ( approximately 60%) when compared to SA ( approximately 40%) immunized mice. Immunization with SA by itself resulted in a mixed Th1/Th2 response whereas co-administration of SA with CpG-ODN resulted in a strong Th1 promoting isotype as they together promoted production of immunoglobulin G2a. Leishmania-specific Th1 cytokine response was induced by co-administering CpG-ODN and SA as they together promoted production of IFN-gamma and IL-12. In the present study, we demonstrate that immunostimulatory phosphorothioate-modified ODN are promising immune enhancers for vaccination against visceral leishmaniaisis.     

Inactivated vaccine induces protection against Mycoplasma agalactiae infection in sheep

The efficacy of an inactivated oil-emulsion vaccine against Mycoplasma agalactiae was evaluated by an experimental infection of sheep. The vaccinated sheep developed high levels of antibodies and, following challenge, they did not develop any clinical signs of disease and the mycoplasmas were not detected, either by isolation trials or PCR assays carried out both on nasal swabs and milk specimens. The unvaccinated-challenged sheep showed typical signs of M. agalactiae infection and bacterial shedding. The results obtained indicate a good efficacy of the vaccine in eliciting protection against M. agalactiae infection.     

Plasmodium falciparum: characterization of a 33-kDa soluble antigen

A 33-kDa soluble antigen identified in the culture supernatant by patient serum and monoclonal antibodies was present in rings, trophozoites, schizonts, and merozoites of Plasmodium falciparum. The antigen which is released into the culture supernatant by growing parasites was also observed in the host cells of trophozoites and schizonts and could be localized on the host cell surface. Its specificity for the surface of trophozoites and schizonts was observed to decrease with increased duration without subculture. The antigen could then be detected on the surface of noninfected erythrocytes. The antigenicity of the 33-kDa antigen was destroyed by heating at 65 degrees C. Monoclonal and polyclonal specific antibodies weakly inhibited parasite growth in vitro. The antigen was present in both knob positive and knob negative parasites in all the P. falciparum isolates tested.     

Partial protection against Eimeria acervulina and Eimeria tenella induced by synthetic peptide vaccine

Coccidiosis is a major parasitic disease of poultry industry and an ideal vaccine should induce long-lasting cross-species protective immunity. Broiler chickens (Cobb 500) were inoculated with single, double or triple injections of a synthetic peptide (derived from sequences of Eimeria acervulina and Eimeria tenella antigens) homogenized in Freund's complete and incomplete adjuvants. The immune responses to the vaccine were assessed by evaluation of antibody and lymphocyte proliferation responses, and the degree of resistance of vaccinated chickens to challenge with sporulated oocysts of E. acervulina or E. tenella determined by comparison of their oocyst output with those of control chickens. The results indicated that the synthetic peptide vaccine induced a high level of antibody and cellular responses associated with partial cross-species protection against challenge with sporulated oocysts of E. acervulina or E. tenella.     

BCG induces protection against Mycobacterium tuberculosis infection in the Wistar rat model

Our understanding of the correlation of Mycobacterium bovis Bacille Calmette-Guerin (BCG)-mediated immune responses and protection against Mycobacterium tuberculosis (Mtb) infection is still limited. We have recently characterized a Wistar rat model of experimental tuberculosis (TB). In the present study, we evaluated the efficacy of BCG vaccination in this model. Upon Mtb challenge, BCG vaccinated rats controlled growth of the bacilli earlier than unvaccinated rats. Histopathology analysis of infected lungs demonstrated a reduced number of granulomatous lesions and lower parenchymal inflammation in vaccinated animals. Vaccine-mediated protection correlated with the rapid accumulation of antigen specific CD4(+) and CD8(+) T cells in the infected lungs. Immunohistochemistry further revealed higher number of CD8(+) cells in the pulmonary granulomas of vaccinated animals. Evaluation of pulmonary immune responses in vaccinated and Mtb infected rats by real time PCR at day 15 post-challenge showed reduced expression of genes responsible for negative regulation of Th1 immune responses. Thus, early protection observed in BCG vaccinated rats correlated with a similarly timed shift of immunity towards the Th1 type response. Our data support the importance of (i) the Th1-Th2 balance in the control of mycobacterial infection and (ii) the value of the Wistar rats in understanding the biology of TB.     

Filarial infection induces protection against P. berghei liver stages in mice

Chronic helminth infections such as filariasis in human hosts can be life long, since parasites are equipped with a repertoire of immune evasion strategies. In many areas where helminths are prevalent, other infections such as malaria are co-endemic. It is still an ongoing debate, how one parasite alters immune responses against another. To dissect the relationships between two different parasites residing in the same host, we established a murine model of co-infection with the filarial nematode Litomosoides sigmodontis and the malaria parasite Plasmodium berghei (ANKA strain). We found that filarial infection of BALB/c mice leads to protection against a subsequent P. berghei sporozoite infection in one-third of co-infected mice, which did not develop blood-stage malaria. This finding did not correlate with adult worm loads, however it did correlate with the presence of microfilariae in blood. Interestingly, protection was abrogated in IL-10-deficient mice. Thus, murine filariasis, in particular when it is a patent infection, is able to modify the immunological balance to induce protection against an otherwise deadly Plasmodium infection and is therefore able to influence the course of malaria in favour of the host.     

Dimethyl itaconate induces long-term innate immune responses and confers protection against infection

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Oligomerization of soluble Fas antigen induces its cytotoxicity

Soluble Fas antigen can protect cells against Fas-mediated apoptosis. High level soluble Fas antigen characteristic for blood of patients with autoimmune disease or cancer is believed to prevent the elimination of autoimmune lymphocytes or tumor cells. Here we first report that human recombinant FasDeltaTM, i.e. soluble Fas generated by alternative splicing of the intact exon 6, is capable of inducing death of transformed cells by "reverse" apoptotic signaling via transmembrane Fas ligand. FasDeltaTM, as well as transmembrane Fas antigen, can be either monomeric or oligomeric, and both its forms are efficient in blocking Fas-mediated apoptosis, although the cytotoxic activity is exhibited solely by the latter. An in vivo analysis of soluble Fas antigen showed that unlike in healthy controls, nearly the total FasDeltaTM present in sera of rheumatoid arthritis patients was oligomeric. This resulted in suppression of cell proliferation in the experimental sera and in its promotion in controls. Thus, oligomerization/depolymerization of soluble Fas antigen can regulate its activity and contribute to the pathogenesis of autoimmune diseases and cancer.     

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.     

Detection of Babesia caballi infection by enzyme-linked immunosorbent assay using recombinant 48-kDa merozoite rhoptry protein

The 48-kDa Babesia caballi merozoite rhoptry protein was expressed using a pGEX4T expression vector in Escherichia coli as glutathione S-transferase fusion protein (GST-BC48), and the expressed GST-BC48 was used in an ELISA to detect specific antibodies in serum samples. No cross-reaction was observed with sera from horses experimentally infected with Babesia equi. GST-BC48 ELISA was a highly sensitive and specific test when compared with the CFT. A total of 209 horse sera obtained from Central Mongolia were examined with the GST-BC48 ELISA and 46.4% (97/209) were found to be seropositive for B. caballi, suggesting that the GST-BC48 ELISA can be successfully used for both quarantine and epidemiological studies.     

Capsular protection against virulent coliphage infection

The ecological significance of bacterial capsules when virulent bacteriophages are present was explored by exposing continuous cultures of Escherichia coli ATCC 11303, in various stages of capsulation and clumping, to a virulent coliphage, T2. Only partial protection was provided by capsulation, but this could be a factor affecting survival in complex mixed cultures.     

The human immune response against the major merozoite surface antigen of Plasmodium falciparum.

The major surface antigen of the merozoite (MMSA) is very immunogenic in humans and it is considered a candidate for developing a malaria vaccine. This protein consists of conserved, dimorphic and polymorphic sequences that might differ in their ability to induce immunity. Epidemiological studies were undertaken in two different endemic areas of West Africa with the aim to identify the sequences within the protein that are the target of the humoral and cellular immune responses. Recombinant polypeptides expressed in E. coli, covering the conserved, the dimorphic and polymorphic regions, were used to evaluate the reactivity of sera and of Peripheral Blood Mononuclear Cells (PBMC) from inhabitants of rural communities exposed to P. falciparum transmission. The analysis of the humoral immune response against the MMSA showed that both qualitative and quantitative differences exist among groups of individuals with different susceptibility to P. falciparum infection. Furthermore, an association between intensity of transmission and antibody reactivity against the dimorphic regions was observed in individuals living in a malaria endemic area. The proliferative response of the PBMC was in most cases very low, however, several T cell clones could be established. The dimorphic region of MMSA was shown to contain T cell epitopes together with sequences most frequently recognized by human sera.     

Methyl jasmonate alters polyamine metabolism and induces systemic protection against powdery mildew infection in barley seedlings

Treatment of the first leaves of barley (Hordeum vulgare L. cv. Golden Promise) seedlings with methyl jasmonate (MJ) led to small, but significant increases in levels of free putrescine and spermine 1 d later and to significant increases in levels of free putrescine, spermidine and spermine by 4 d following treatment. MJ-treated first leaves also exhibited significant increases in the amounts of soluble conjugates of putrescine and spermidine 1, 2 and 4 d after treatment. In second leaves of plants where the first leaves had been treated with MJ, no significant changes in levels of free polyamines were observed, but significant increases in levels of soluble conjugates of putrescine and spermidine were detected. These changes were accompanied by increased activities of soluble ornithine decarboxylase (ODC), soluble and particulate arginine decarboxylase (ADC), and S-adenosylmethionine decarboxylase (AdoMetDC), in first and second leaves following treatment of the first leaves with MJ. Activities of soluble and particulate diamine oxidase (DAO) were also higher in first and second leaves following treatment of the first leaves with MJ. Treatment of the first leaves with MJ led to a significant reduction in powdery mildew (Blumeria graminis f. sp. hordei) infection on the second leaves and also resulted in significant increases in activities of the plant defence-related enzymes, phenylalanine ammonia lyase (PAL) and peroxidase.     

Development of CRISPR-CAS9 based RNA drugs against Eimeria tenella infection

Parasitic diseases are major trouble in many parts of the world. We consider that if a chemical can break a DNA barcode sequence, it might be used to develop a species-specific anti-parasitic agent. To examine this hypothesis, we constructed sgRNAs that target both the control (5.8S rDNA) and a DNA barcode (ITS) sequence in Eimeria tenella. In vitro experiment showed that Cas9 mRNA combined with sgRNAs could reduce the sporulation percentage of oocysts and the survival rate of sporulated oocysts and sporozoites. Quantitative real-time PCR showed that the DNAs of parasites exposed to Cas9 mRNA and sgRNAs were significantly affected, regardless of whether they were exposed to a combination of two sgRNAs or just a single sgRNA. The DNA sequencing also indicated that the experimental group exposed to two sgRNAs mixed with Cas9-induced deletion of large parts and a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments. In vivo trial, the effect of sgRNA and Cas9 RNA on the pathogenicity of E. tenella in chicken showed less lesion score and oocysts score (P < 0.05) in experimental groups than control groups. The results and concepts presented in this research can lead to discovering novel nucleic acid therapeutic drugs for Eimeriasis and other parasitic infections, which provide insights into the development of species-specific anti-parasitic agents.     

Bile acid-independent protection against Clostridioides difficile infection

Clostridioides difficile infections occur upon ecological / metabolic disruptions to the normal colonic microbiota, commonly due to broad-spectrum antibiotic use. Metabolism of bile acids through a 7α-dehydroxylation pathway found in select members of the healthy microbiota is regarded to be the protective mechanism by which C. difficile is excluded. These 7α-dehydroxylated secondary bile acids are highly toxic to C. difficile vegetative growth, and antibiotic treatment abolishes the bacteria that perform this metabolism. However, the data that supports the hypothesis that secondary bile acids protect against C. difficile infection is supported only by in vitro data and correlative studies. Here we show that bacteria that 7α-dehydroxylate primary bile acids protect against C. difficile infection in a bile acid-independent manner. We monoassociated germ-free, wildtype or Cyp8b1^-/- (cholic acid-deficient) mutant mice and infected them with C. difficile spores. We show that 7α-dehydroxylation (i.e., secondary bile acid generation) is dispensable for protection against C. difficile infection and provide evidence that Stickland metabolism by these organisms consumes nutrients essential for C. difficile growth. Our findings indicate secondary bile acid production by the microbiome is a useful biomarker for a C. difficile-resistant environment but the microbiome protects against C. difficile infection in bile acid-independent mechanisms.     

Apoptotic deletion of Th cells specific for the 19-kDa carboxyl-terminal fragment of merozoite surface protein 1 during malaria infection

Immunity induced by the 19-kDa fragment of merozoite surface protein 1 is dependent on CD4+ Th cells. However, we found that adoptively transferred CFSE-labeled Th cells specific for an epitope on Plasmodium yoelii 19-kDa fragment of merozoite surface protein 1 (peptide (p)24), but not OVA-specific T cells, were deleted as a result of P. yoelii infection. As a result of infection, spleen cells recovered from infected p24-specific T cell-transfused mice demonstrated reduced response to specific Ag. A higher percentage of CFSE-labeled p24-specific T cells stained positive with annexin and anti-active caspase-3 in infected compared with uninfected mice, suggesting that apoptosis contributed to deletion of p24-specific T cells during infection. Apoptosis correlated with increased percentages of p24-specific T cells that stained positive for Fas from infected mice, suggesting that P. yoelii-induced apoptosis is, at least in part, mediated by Fas. However, bystander cells of other specificities also showed increased Fas expression during infection, suggesting that Fas expression alone is not sufficient for apoptosis. These data have implications for the development of immunity in the face of endemic parasite exposure.