Intranasal administration of the synthetic polypeptide from the C-terminus of the circumsporozoite protein of Plasmodium berghei with the modified heat-labile toxin of Escherichia coli (LTK63) induces a complete protection against malaria challenge.

Needle-free procedures are very attractive ways to deliver vaccines because they diminish the risk of contamination and may reduce local reactions, pain or pain fear especially in young children with a consequence of increasing the vaccination coverage for the whole population. For this purpose, the possible development of a mucosal malaria vaccine was investigated. Intranasal immunization was performed in BALB/c mice using a well-studied Plasmodium berghei model antigen derived from the circumsporozoite protein with the modified heat-labile toxin of Escherichia coli (LTK63), which is devoid of any enzymatic activity compared to the wild type form. Here, we show that intranasal administration of the two compounds activates the T and B cell immune response locally and systemically. In addition, a total protection of mice is obtained upon a challenge with live sporozoites.

Randomized double-blind controlled Phase I/IIa trial to assess the efficacy of malaria vaccine PfCS102 to protect against challenge with P. falciparum.

The aim of this Phase I/IIa double-blind controlled trial was to test the efficacy of the sporozoite-based malaria vaccine PfCS 282-383 (PfCS102) to protect against Plasmodium falciparum parasitaemia. 16 volunteers were randomized to receive twice 30 μg of PfCS102 formulated in Montanide ISA 720 or ISA 720 alone (control). Two weeks after 2nd immunization, volunteers were challenged using 5 infected mosquitoes. All vaccinees developed antibodies against PfCS102 versus none control. 8/8 vaccinees and 6/6 controls challenged developed malaria parasitaemia. The duration from infection to onset of patent parasitaemia was similar in both groups (214 h in vaccinees and 216 in controls). PfCS102 is safe and immunogenic but provides no protection against artificial challenge in its current formulation.

From pathogen defence to food allergy; The multiple facets of the mucosal immune system

Résumé destiné à un large public Le système immunitaire associé aux muqueuses gastro-intestinales doit être capable de protéger notre organisme contre l'invasion de pathogènes. Parallèlement, il doit identifier en Cant que tels, des composés inoffensifs comme la nourriture ou les milliards de bactéries qui résident dans notre intestin. Le travail présenté ici aborde ces deux aspects essentiels au bon fonctionnement de notre muqueuse intestinale. Dans une première partie, la protéine nommée pièce sécrétoire a été étudiée pour ses propriétés protectrices contre le pathogène viral rotavirus. Le rôle de la pièce sécrétoire est de transporter les anticorps que nous produisons vers la surface des muqueuses. En dehors de cette fonction bien connue, il se peut que cette protéine soit également capable de protéger notre organisme contre certains virus. L'hypothèse de travail était donc que la pièce sécrétoire se lie directement au virus, l'empêchant ainsi d'infecter des cellules épithéliales de l'intestin. En utilisant différentes techniques biochimiques, cette hypothèse s'est révélée fausse car aucune interaction entre la pièce sécrétoire et le virus n'a pu être observée, et logiquement, aucune protection n'a pu prendre place. En revanche, la pièce sécrétoire se lie à d'autres structures pathogéniques et permet ainsi de neutraliser leurs effets néfastes. La pièce sécrétoire participe donc activement à la protection de nos muqueuses, en plus de son rôle de transporteur. La deuxième partie de ce travail avait pour sujet les réactions inappropriées que le système immunitaire induit parfois contre un aliment, ou, autrement dit, les allergies alimentaires. Un modèle d'allergie alimentaire à donc été développé chez la souris et a permis de mesurer plusieurs symptômes et facteurs liés à l'allergie. Puis, ce modèle a été utilisé afin de tester les effets bénéfiques d'une bactérie lactique, dite probiotique, sur le développement de l'allergie. Il a été observé que, sous certaines circonstances, l'administration de la bactérie lactique protégeait entièrement les souris contre les réactions allergiques. L'effet bénéfique dépend donc du probiotique mais également d'autres facteurs encore inconnus â ce jour. Cette étude ouvre la voie sur la compréhension des mécanismes liés aux allergies alimentaires et sur l'impact que peuvent avoir les bactéries probiotiques sur cette maladie. Résumé Le système immunitaire associé aux muqueuses intestinales doit être capable de différencier les antigènes inoffensifs tels que 1a nourriture ou les bactéries commensales des microorganismes potentiellement dangereux. Cet aspect est essentiel pour le maintien de l'homéostase intestinale et fait l'objet du travail présenté ici. Dans un premier projet, les propriétés protectrices de la protéine appelée pièce sécrétoire (SC) ont été étudiées. SC est une protéine connue pour le transport des immunoglobulines à la surface des muqueuses. Cette protéine est fortement glycosylée paz des sucres complexes, ce qui nous a mené à postuler que SC puisse interagir avec le pathogène rotavirus. Cette hypothèse était soutenue par le fait que ce virus adhère aux cellules épithéliales par des résidus glycosylés. Des analyses biochimiques et biologiques ont démontré qu'aucune interaction entre SC et le virus ne prenait place, et que par conséquent SC n'offrait aucune protection contre ce pathogène. En revanche, SC interagit avec d'autres structures pathogéniques, comme la toxine A de Clostridium difficile, et la molécule d'adhésion intimine de la bactérie entéropathogène Escherichia coli. La liaison se fait par l'intermédiaire des sucres et confère ainsi une protection contre ces pathogènes. Ainsi, SC a été identifié comme agent neutralisant au niveau de l'intestin. La deuxième partie de ce travail abordait le sujet des allergies alimentaires, et avait pour but de tester les effets bénéfiques potentiels d'une bactérie probiotique, Lactobacillus paracasei NCC2461, contre les réactions allergiques. Un modèle marin d'allergie alimentaire a été mis au point, permettant de mesurer des immunoglobulines E, des symptômes allergiques, et la dégranulation de mastocytes. Lorsque le probiotique a été administré aux souris, celles-ci ont été complètement protégées des réactions allergiques dans une première expérience. Cependant, cette protection n'a pas été reproduite et suggère que des facteurs environnementaux encore inconnus sont critiques pour que le probiotique agisse positivement. Ce travail a permis de mettre en évidence la complexité de l'approche des traitements liés aux probiotiques et ouvre la voie sur la compréhension des mécanismes liés à l'allergie. Abstract The mucosal immune system associated to the gastrointestinal mucosa must efficiently distinguish between innocuous antigens, such as food proteins and commensal bacteria and potentially infectious agents. The work presented here deals with these two essential aspects guaranteeing intestinal homeostasis. In the first part of this work, the protective properties of secretory component (SC) toward the pathogen rotavirus were investigated. SC, which allows the transport of polymeric immunoglobulins (Ig) to mucosal surfaces, is highly glycosylated with complex glycan structures. The abundance and the nature of these carbohydrates led us to speculate that SC might interact with rotavirus, which is known to bind target cells with glycan receptors. Using various biological and biochemical techniques, we demonstrated that SC did not interact with rotaviruses, nor protected epithelial cells from infection. However, SC was shown to bind to Clostridium difficile toxin A and to the enteropathogenic Echerischia coli adhesion molecule intimin in a glycan-dependent fashion. These interactions allow in vitro protection of epithelial cells using physiological concentrations of SC. These data identify SC as a microbial scavenger at mucosal surfaces, and in the context of secretory IgA, further enhance the neutralising properties of the complex. The second project was inscribed in the domain of food allergy and aimed to test the modulatory functions of a probiotic strain of Lactobacillus paracasei toward allergic reactions. A model of food-mediated allergy was developed in the mouse using mucosal sensitisation. Several parameters associated to allergy were quantified after allergen challenge, and included allergen-specific IgE, allergic signs like diarrhea and temperature drop, and degranulation of mast cells. Administration of the probiotic strain was shown to completely protect mice from allergic reactions. However, these data were not reproduced, suggesting that unknown environmental factors are required so that protection mediated by the probiotic strain occurs. This study paves the way to the understanding of the mechanisms associated to allergy, and highlights the tremendous complexity that probiotic treatments will have to face.

Immune Antibodies and Helminth Products Drive CXCR2-Dependent Macrophage-Myofibroblast Crosstalk to Promote Intestinal Repair.

Helminth parasites can cause considerable damage when migrating through host tissues, thus making rapid tissue repair imperative to prevent bleeding and bacterial dissemination particularly during enteric infection. However, how protective type 2 responses targeted against these tissue-disruptive multicellular parasites might contribute to homeostatic wound healing in the intestine has remained unclear. Here, we observed that mice lacking antibodies (Aid-/-) or activating Fc receptors (Fcrg-/-) displayed impaired intestinal repair following infection with the murine helminth Heligmosomoides polygyrus bakeri (Hpb), whilst transfer of immune serum could partially restore chemokine production and rescue wound healing in Aid-/- mice. Impaired healing was associated with a reduced expression of CXCR2 ligands (CXCL2/3) by macrophages (MΦ) and myofibroblasts (MF) within intestinal lesions. Whilst antibodies and helminths together triggered CXCL2 production by MΦ in vitro via surface FcR engagement, chemokine secretion by intestinal MF was elicited by helminths directly via Fcrg-chain/dectin2 signaling. Blockade of CXCR2 during Hpb challenge infection reproduced the delayed wound repair observed in helminth infected Aid-/- and Fcrg-/- mice. Finally, conditioned media from human MΦ stimulated with infective larvae of the helminth Ascaris suum together with immune serum, promoted CXCR2-dependent scratch wound closure by human MF in vitro. Collectively our findings suggest that helminths and antibodies instruct a chemokine driven MΦ-MF crosstalk to promote intestinal repair, a capacity that may be harnessed in clinical settings of impaired wound healing.

Plant Immune Responses: Aphids Strike Back.

To survive and complete their life cycle, herbivorous insects face the difficult challenge of coping with the arsenal of plant defences. A new study reports that aphids secrete evolutionarily conserved cytokines in their saliva to suppress host immune responses.

Plant Immune Responses: Aphids Strike Back.

To survive and complete their life cycle, herbivorous insects face the difficult challenge of coping with the arsenal of plant defences. A new study reports that aphids secrete evolutionarily conserved cytokines in their saliva to suppress host immune responses.

Immune correlates of vaccine protection against HIV-1 acquisition.

The partial efficacy reported in the RV144 HIV vaccine trial in 2009 has driven the HIV vaccine field to define correlates of risk associated with HIV-1 acquisition and connect these functionally to preventing HIV infection. Immunological correlates, mainly including CD4(+) T cell responses to the HIV envelope and Fc-mediated antibody effector function, have been connected to reduced acquisition. These immunological correlates place immunological and genetic pressure on the virus. Indeed, antibodies directed at conserved regions of the V1V2 loop and antibodies that mediate antibody-dependent cellular cytotoxicity to HIV envelope in the absence of inhibiting serum immunoglobulin A antibodies correlated with decreased HIV risk. More recently, researchers have expanded their search with nonhuman primate studies using vaccine regimens that differ from that used in RV144; these studies indicate that non-neutralizing antibodies are associated with protection from experimental lentivirus challenge as well. These immunological correlates have provided the basis for the design of a next generation of vaccine regimens to improve upon the qualitative and quantitative degree of magnitude of these immune responses on HIV acquisition.

Genital immunization of heifers with a glycoprotein Edeleted, recombinant bovine herpesvirus 1 strain confers protection upon challenge with a virulent isolate

Venereal infection of seronegative heifers and cows with bovine herpesvirus type 1.2 (BoHV-1.2) frequently results in vulvovaginitis and transient infertility. Parenteral immunization with inactivated or modified live BoHV-1 vaccines often fails in conferring protection upon genital challenge. We herein report an evaluation of the immune response and protection conferred by genital vaccination of heifers with a glycoprotein E-deleted recombinant virus (SV265gE-). A group of six seronegative heifers was vaccinated with SV265gE- (0,2mL containing 10(6.9)TCID50) in the vulva submucosa (group IV); four heifers were vaccinated intramuscularly (group IM, 1mL containing 10(7.6)TCID50) and four heifers remained as non-vaccinated controls. Heifers vaccinated IV developed mild, transient local edema and hyperemia and shed low amounts of virus for a few days after vaccination, yet a sentinel heifer maintained in close contact did not seroconvert. Attempts to reactivate the vaccine virus in two IV vaccinated heifers by intravenous administration of dexamethasone (0.5mg/kg) at day 70 pv failed since no virus shedding, recrudescence of genital signs or seroconversion were observed. At day 70 pv, all vaccinated and control heifers were challenged by genital inoculation of a highly virulent BoHV-1.2 isolate (SV56/90, 10(7.1)TCID50/animal). After challenge, virus shedding was detected in genital secretions of control animals for 8.2 days (8-9); in the IM group for 6.2 days (4-8 days) and during 5.2 days (5-6 days) in the IV group. Control non-vaccinated heifers developed moderate (2/4) or severe (2/4) vulvovaginitis lasting 9 to 13 days (x: 10.7 days). The disease was characterized by vulvar edema, vulvo-vestibular congestion, vesicles progressing to coalescence and erosions, fibrino-necrotic plaques and fibrinopurulent exudate. IM vaccinated heifers developed mild (1/3) or moderate (3/4) genital lesions, lasting 10 to 12 days (x: 10.7 days); and IV vaccinated heifers developed mild and transient vulvovaginitis (3/4) or mild to moderate genital lesions (1/4). In the IV group, the clinical signs lasted 4 to 8 days (x: 5.5 days). Clinical examination of the animals after challenge revealed that vaccination by both routes conferred some degree of protection, yet IV vaccination was clearly more effective in reducing the severity and duration of clinical disease. Furthermore, IV vaccination reduced the period of virus shedding in comparison with both groups. Taken together, these results demonstrate that SV265gE- is sufficiently attenuated upon IV vaccination in a low-titer dosis, is not readily reactivated after corticosteroid treatment and lastly, and more importantly, confers local protection upon challenge with a high titer of a virulent heterologous BoHV-1 isolate. Therefore, the use of this recombinant for genital immunization may be considered for prevention of BoHV-1-associated genital disease in the field.

Induction of immune response in broiler chickens immunized with recombinant FliC and challenged by Salmonella Typhimurium

The study examined (1) the immune response in broiler chickens after oral immunization with recombinant flagellin (rFliC) from Salmonella Typhimurium conjugated with sodium alginate microparticles, and the immune response enhancement in association with recombinant cholera toxin B subunit protein (rCTB) and pool of Lactobacillus spp. (PL). The immune responses were evaluated by dosage of IgY serum and IgA from intestinal fluid and immunostaining of CD8+ T lymphocytes in the cecum. The immunized animals were challenged with Salmonella Typhimurium (ST) 21 days after treatment. In all immunized groups, a significant increase (p<0.05) was observed in IgA levels (μg/mL), especially three weeks after immunization. The serum IgY levels (μg/mL) were little affected by the treatments and differed significantly among groups only in the second post-immunization week (p<0.05). After the challenge, the number of CD8+ T cells differed significantly between the treatments and negative control. Retrieval of Salmonella Typhimurium was not detected at 48 hours after the challenge in T2 (rFliC+rCTb), T3 (rFliC+PL) and T4 (rFliC+rCTB PL). The rFliC administered orally with or without rCTB and Lactobacillus spp. produces significant induction of humoral immune response, and the immunized chickens were more effective in eliminating Salmonella after challenge.

A single strain of Mycobacterium bovis bacillus Calmette-Guérin (BCG) grown in two different media evokes distinct humoral immune responses in mice

Two attenuated bacillus Calmette-Guérin (BCG) preparations derived from the same Moreau strain, Copenhagen but grown in Sauton medium containing starch and bacto-peptone (onco BCG, O-BCG), or asparagine (intradermal BCG, ID-BCG), exhibited indistinguishable DNA sequences and bacterial morphology. The number of viable bacilli recovered from spleen, liver and lungs was approximately the same in mice inoculated with the vaccines and was similarly reduced (over 90%) in mice previously immunized with either BCG vaccine. The humoral immune response evoked by the vaccines was, however, distinct. Spleen cell proliferation accompanying the growth of bacilli in tissue was significantly higher in mice inoculated with O-BCG. These cells proliferated in vitro upon challenge with the corresponding BCG extract. Previous cell treatment with mAb anti-CD4 T cells abolished this effect. Anti-BCG antibodies, as assayed either in serum by ELISA or by determining the number of antibody-producing spleen cells by the spot-ELISA method, were significantly higher in mice inoculated with ID-BCG. Anti-BCG antibodies were detected in all immunoglobulin classes, but they were more prevalent in IgG with the following distribution among its isotypes: IgG1>(IgG2a = IgG2b)>IgG3. When some well-characterized Mycobacterium tuberculosis antigens were used as substitutes for BCG extracts in ELISA, although antibodies against the 65-kDa and 96-kDa proteins were detected significantly, antibodies against the 71-kDa, 38-kDa proteins and lipoarabinomannan were only barely detected or even absent. These results indicate that BCG bacilli cultured in Sauton-asparagine medium permitted the multiplication of bacilli, tending to induce a stronger humoral immune response as compared with bacilli grown in Sauton-starch/bacto-peptone-enriched medium.

Recombinant E2 glycoprotein of bovine viral diarrhea virus induces a solid humoral neutralizing immune response but fails to confer total protection in cattle

Two recombinant baculoviruses were produced in order to obtain a bovine viral diarrhea virus (BVDV) immunogen: AcNPV/E2 expressing E2 glycoprotein, and AcNPV/E0E1E2 expressing the polyprotein region coding for the three structural proteins of BVDV (E0, E1, and E2). Mice were immunized with Sf9 cells infected with the recombinant baculoviruses in a water in oil formulation and the production of neutralizing antibodies was evaluated. Since E2 elicited higher neutralizing antibody titers than E0-E1-E2 polyprotein, it was selected to immunize cattle. Calves received two doses of recombinant E2 vaccine and were challenged with homologous BVDV 37 days later. The recombinant immunogen induced neutralizing titers which showed a mean value of 1.5 ± 0.27 on the day of challenge and reached a top value of 3.36 ± 0.36, 47 days later (84 days post-vaccination). On the other hand, sera from animals which received mock-infected Sf9 cells did not show neutralizing activity until 25 days post-challenge (62 days post-vaccination), suggesting that these antibodies were produced as a consequence of BVDV challenge. Even when no total protection was observed in cattle, in vitro viral neutralization assays revealed that the recombinant immunogen was able to induce neutralizing antibody synthesis against the homologous strain as well as against heterologous strains in a very efficient way.

Cocaine/levamisole-induced systemic vasculitis with retiform purpura and pauci-immune glomerulonephritis

Levamisole has been increasingly used as an adulterant of cocaine in recent years, emerging as a public health challenge worldwide. Levamisole-associated toxicity manifests clinically as a systemic vasculitis, consisting of cutaneous, hematological, and renal lesions, among others. Purpura retiform, cutaneous necrosis, intravascular thrombosis, neutropenia, and less commonly crescentic nephritis have been described in association with anti-neutrophil cytoplasmic antibodies (ANCAs) and other autoantibodies. Here we report the case of a 49-year-old male who was a chronic cocaine user, and who presented spontaneous weight loss, arthralgia, and 3 weeks before admission purpuric skin lesions in the earlobes and in the anterior thighs. His laboratory tests on admission showed serum creatinine of 4.56 mg/dL, white blood count 3,800/μL, hemoglobin 7.3 g/dL, urinalysis with 51 white blood cells/μL and 960 red blood cells/μL, and urine protein-to-creatinine ratio 1.20. Serum ANCA testing was positive (>1:320), as well as serum anti-myeloperoxidase and anti-proteinase 3 antibodies. Urine toxicology screen was positive for cocaine and levamisole, with 62.8% of cocaine, 32.2% of levamisole, and 5% of an unidentified substance. Skin and renal biopsies were diagnostic for leukocytoclastic vasculitis and pauci-immune crescentic glomerulonephritis, respectively. The patient showed a good clinical response to cocaine abstinence, and use of corticosteroids and intravenous cyclophosphamide. Last serum creatinine was 1.97 mg/dL, white blood cell count 7,420/μL, and hemoglobin level 10.8 g/dL. In levamisole-induced systemic vasculitis, the early institution of cocaine abstinence, concomitant with the use of immunosuppressive drugs in severe cases, may prevent permanent end organ damage and associate with better clinical outcomes.

Molecular characterization of a crustinlike antimicrobial peptide in the giant tiger shrimp, Penaeus monodon, and its expression profile in response to various immunostimulants and challenge with WSSV

A crustinlike antimicrobial peptide from the haemocytes of giant tiger shrimp, Penaeus monodon was partially characterized at the molecular level and phylogenetic analysis was performed. The partial coding sequence of 299 bp and 91 deduced amino acid residues possessed conserved cysteine residues characteristic of the shrimp crustins. Phylogenetic tree and sequence comparison clearly confirmed divergence of this crustinlike AMP from other shrimp crustins. The differential expression of the crustinlike AMP in P. monodon in response to the administration of various immunostimulants viz., two marine yeasts (Candida haemulonii S27 and Candida sake S165) and two bglucan isolates (extracted from C. haemulonii S27 and C. sake S165) were noted during the study. Responses to the application of two grampositive probiotic bacteria (Bacillus MCCB101 and Micrococcus MCCB104) were also observed. The immune profile was recorded preand postchallenge white spot syndrome virus (WSSV) by semiquantitative RTPCR. Expressions of seven WSSV genes were also observed for studying the intensity of viral infection in the experimental animals. The crustinlike AMP was found to be constitutively expressed in the animal and a significant downregulation could be noted postchallenge WSSV. Remarkable downregulation of the gene was observed in the immunostimulant fed animals prechallenge followed by a significant upregulation postchallenge WSSV. Tissuewise expression of crustinlike AMP on administration of C. haemulonii and Bacillus showed maximum transcripts in gill and intestine. The marine yeast, C. haemulonii and the probiotic bacteria, Bacillus were found to enhance the production of crustinlike AMP and confer significant protection to P. monodon against WSSV infection

Litomosoides sigmodontis: vaccine-induced immune responses against Wolbachia surface protein can enhance the survival of filarial nematodes during primary infection

Wolbachia are bacteria present within the tissues of most filarial nematodes. Filarial nematode survival is known to be affected by immune responses generated during filarial nematode infection and immune responses to Wolbachia can be found in different species harbouring filarial nematode infections, including humans. Using the rodent filarial model Litomosoides sigmodontis, we show that pre-exposure to wolbachia surface protein in a Th1 context (but not in a Th2-context) enhances worm survival on subsequent challenge. This study suggests that despite abundant evidence that pro-inflammatory reactions to the endosymbiont have detrimental effects on the both the nematode and mammalian host, they may under some circumstances be beneficial to the nematode.

The expression of ferritin, lactoferrin, transferrin receptor and solute carrier family 11A1 in the host response to BCG-vaccination and Mycobacterium tuberculosis challenge

Iron is an essential cofactor for both mycobacterial growth during infection and for a successful protective immune response by the host. The immune response partly depends on the regulation of iron by the host, including the tight control of expression of the iron-storage protein, ferritin. BCG vaccination can protect against disease following Mycobacterium tuberculosis infection, but the mechanisms of protection remain unclear. To further explore these mechanisms, splenocytes from BCG-vaccinated guinea pigs were stimulated ex vivo with purified protein derivative from M. tuberculosis and a significant down-regulation of ferritin light- and heavy-chain was measured by reverse-transcription quantitative-PCR (P ≤0.05 and ≤0.01, respectively). The mechanisms of this down-regulation were shown to involve TNFα and nitric oxide. A more in depth analysis of the mRNA expression profiles, including genes involved in iron metabolism, was performed using a guinea pig specific immunological microarray following ex vivo infection with M. tuberculosis of splenocytes from BCG-vaccinated and naïve guinea pigs. M. tuberculosis infection induced a pro-inflammatory response in splenocytes from both groups, resulting in down-regulation of ferritin (P ≤0.05). In addition, lactoferrin (P ≤0.002), transferrin receptor (P ≤0.05) and solute carrier family 11A1 (P ≤0.05), were only significantly down-regulated after infection of the splenocytes from BCG-vaccinated animals. The results show that expression of iron-metabolism genes is tightly regulated as part of the host response to M. tuberculosis infection and that BCG-vaccination enhances the ability of the host to mount an iron-restriction response which may in turn help to combat invasion by mycobacteria.