Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/ disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290-307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.

Prevalence of Human T-Cell Lymphotropic Virus (HTLV-1/2) in Individuals from Public Health Centers in Mozambique

The prevalence of human T-cell lymphotropic viruses types 1 and 2 (HTLV-1/2) in Mozambique is not known. The present study examined blood samples from 208, 226, and 318 individuals from Northern, Central, and Southern Mozambique, respectively, of all socioeconomic and demographic strata attending public health centers in Mozambique for HTLV-1/2-specific antibodies. Serum samples were assessed for HIV- and HTLV-1/2-specific antibodies by using enzyme immunoassays, and infections with HTLV-1 and -2 were confirmed by using Western blot. An overall HTLV-1/2 prevalence of 2.3% (2.9% in female and 1.1% in male subjects) was observed, and the prevalence of infection increased with age. Regional variation in the prevalence of HIV and HTLV-1/2 was observed; 32.2%, 65.5%, and 44% of individuals tested HIV positive in Northern, Central, and Southern Mozambique, respectively, and 2.4%, 3.9%, and 0.9% tested HTLV-1/2 positive in the same regions. HTLV-1 infection was confirmed in these individuals. No association between HTLV-1 infection and socio-demographic variables or HIV status was detected, although the low number of HTLV-1-positive cases did not allow robust statistical analyses. The results obtained suggest different risk factors and epidemiologic correlates of HIV and HTLV-1 transmission in Mozambique. Furthermore, our results suggested that North and Central Mozambique should be considered endemic regions for HTLV-1 infection. As no cases of HTLV-2 were detected, HTLV-2 appears to have not been introduced into Mozambique.

Mast cell repopulation of the peritoneal cavity: contribution of mast cell progenitors versus bone marrow derived committed mast cell precursors

Background: Mast cells have recently gained new importance as immunoregulatory cells that are involved in numerous pathological processes. One result of these processes is an increase in mast cell numbers at peripheral sites. This study was undertaken to determine the mast cell response in the peritoneal cavity and bone marrow during repopulation of the peritoneal cavity in rats. Results: Two mast cell specific antibodies, mAb AA4 and mAb BGD6, were used to distinguish the committed mast cell precursor from more mature mast cells. The peritoneal cavity was depleted of mast cells using distilled water. Twelve hours after distilled water injection, very immature mast cells could be isolated from the blood and by 48 hours were present in the peritoneal cavity. At this same time the percentage of mast cells in mitosis increased fourfold. Mast cell depletion of the peritoneal cavity also reduced the total number of mast cells in the bone marrow, but increased the number of mast cell committed precursors. Conclusions: In response to mast cell depletion of the peritoneal cavity, a mast cell progenitor is released into the circulation and participates in repopulation of the peritoneal cavity, while the committed mast cell precursor is retained in the bone marrow.

Passive immunization with monoclonal antibody against a 70-kDa putative adhesin of Sporothrix schenckii induces protection in murine sporotrichosis

Cell-mediated and innate immunity are considered the most important mechanisms of host defense against fungus infections. However, recent studies demonstrated that specific antibodies show different degrees of protection against mycosis. In a previous study, antigens secreted by Sporothrix schenckii induced a specific humoral response in infected animals, mainly against the 70-kDa molecule, indicating a possible participation of antibodies to this antigen in infection control. in the present study, an IgG1 mAb was produced against a 70-kDa glycoprotein of S. schenckii in order to better understand the effect of passive immunization of mice infected with S. schenckii. Results showed a significant reduction in the number of CFU in organs of mice when the mAb was injected before and during S. schenckii infection. Similar results were observed when T-cell-deficient mice were used. Moreover, in a second schedule treatment, the mAb was injected after infection was established, and again we observed a significant reduction in CFU associated with an increase of IFN-gamma production. Also, the 70-kDa antigen is shown to be a putative adhesin present on the surface of this fungus. In conclusion, we report for the first time the protective effect of a specific antibody against S. schenckii.

A recombinant vaccine based on domain II of Plasmodium vivax Apical Membrane Antigen 1 induces high antibody titres in mice

The Apical Membrane Antigen 1 (AMA-1) is considered a promising candidate for development of a malaria vaccine against asexual stages of Plasmodium. We recently identified domain II (DII) of Plasmodium vivax AMA-1 (PvAMA-1) as a highly immunogenic region recognised by IgG antibodies present in many individuals during patent infection with P. vivax. The present study was designed to evaluate the immunogenic properties of a bacterial recombinant protein containing PvAMA-1 DII. To accomplish this, the recombinant protein was administered to mice in the presence of each of the following six adjuvants: Complete/Incomplete Freund`s Adjuvant (CFA/IFA), aluminium hydroxide (Alum), Quil A, QS21 saponin, CpG-ODN 1826 and TiterMax. We found that recombinant DII was highly immunogenic in BALB/c mice when administered in the presence of any of the tested adjuvants. Importantly, we show that DII-specific antibodies recognised the native AMA-1 protein expressed on the surface of P. vivax merozoites isolated from the blood of infected patients. These results demonstrate that a recombinant protein containing PvAMA-1 DII is immunogenic when administered in different adjuvant formulations, and indicate that this region of the AMA-1 protein should continue to be evaluated as part of a subunit vaccine against vivax malaria. (C) 2010 Elsevier Ltd. All rights reserved.

Innate immune lectins kill bacteria expressing blood group antigen

The expression of ABO(H) blood group antigens causes deletion of cells that generate self-specific antibodies to these antigens but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against such pathogens, given these limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectin-4 (Gal-4) and Gal-8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing Escherichia coli while failing to alter the viability of other E. coli strains or other Gram-negative or Gram-positive organisms both in vitro and in vivo. The killing activity of both Gal-4 and Gal-8 is mediated by their C-terminal domains, occurs rapidly and independently of complement and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that express blood group-like antigens on their surface.

Expression of Human Recombinant Antibody Fragments Capable of Partially Inhibiting the Phospholypase Activity of Crotalus durissus terrificus Venom

Crotoxin is the main toxic component of the South American rattlesnake Crotalus durissus terrificus venom. It is composed of two different subunits: CA, crotapotin, and CB (basic subunit of cortoxin isolated from C. d. terrificus), a weakly toxic phospholipase A(2) with high enzymatic activity. The phospholipases A(2) are abundant in snake venoms and are responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. However, in addition to their normal digestive action, a wide range of pharmacological activities, such as neurotoxic, myotoxic, oedema-inducing, hypotensive, platelet-aggregating, cardiotoxic, and anticoagulant effects have been attributed to venom phospholipases A(2). In this study, we used a non-immune human single-chain fragment variable library, Griffin.1 (Medical Research Council, Cambridge, UK) for selection of recombinant antibodies against antigens present in C. d. terrificus venom and identification of specific antibodies able to inhibit the phospholipase activity. Two clones were identified as capable of inhibiting partially this activity in vitro. These clones were able to reduce in vivo the myotoxic and oedema-inducing activity of CB and the lethality of C. d. terrificus venom and crotoxin, but had no effect on the in vitro anticoagulant activity of CB. These results demonstrate the potential of using recombinant single-chain fragment variable libraries in the production of antivenoms.

The prospects for immunotherapy in smoking cessation

Context Smoking is a major preventable cause of death and disability that is maintained by dependence on nicotine. Smoking cessation reduces mortality and morbidity. Although existing pharmacological aids to smoking cessation and relapse prevention (nicotine replacement therapy and bupropion) improve on unassisted quitting and behavioural methods, they are only modestly effective. More effective pharmacological methods are required that improve compliance, reduce side-effects, and can be used in combination with existing cessation methods. Starting point A nicotine vaccine is a promising immunotherapeutic approach to smoking cessation and relapse prevention. Such a vaccine would induce the immune system to form specific antibodies to nicotine to prevent it from crossing the blood-brain barrier to act on receptor sites in the central nervous system. Recent studies in rats provide proof of principle by showing that nicotine-specific antibodies can prevent the reinstatement of nicotine self-administration (N Lindblom et al, Respiration 2002; 69: 254–60) and block dopamine release in the shell of the nucleus accumbens (Sde Villiers et al, Respiration 2002; 69: 247–53). A phase 1 trial of a human cocaine vaccine has also recently been successfully completed (T Kosten et al, Vaccine 2002; 20: 1196–204). A safe and effective human nicotine vaccine would potentially have fewer side-effects and better compliance than existing smoking-cessation pharmacotherapies. It could also be used in combination with some of them (eg, bupropion). Where next? The most promising clinical application of a human nicotine vaccine is likely to be in relapse prevention in abstinent smokers. A vaccine may also have a role in preparing smokers to quit. Clinical trials of safety and efficacy in human smokers and ex-smokers are warranted. If a nicotine vaccine proves to be safe and effective, the health-care system will need to ensure that it is registered for clinical use and that the poorer members of the community (among whom smoking prevalence is now highest in developed countries) have access to the vaccine. The community will need to be appropriately informed about the role of a nicotine vaccine to ensure that it is not prematurely used for preventive purposes in children and adolescents.

C4d staining in post-reperfusion renal biopsy is not useful for the early detection of antibody-mediated rejection when CDC crossmatching is negative

Background. Sensitized patients (pts) may develop acute antibody-mediated rejection (AMR) due to preformed donor-specific antibodies, undetected by pre-transplant complement-dependent cytotoxicity (CDC) crossmatch (XM). We hypothesized that C4d staining in 1-h post-reperfusion biopsies (1-h Bx) could detect early complement activation in the renal allograft due to preformed donor-specific antibodies. Methods. To test this hypothesis, renal transplants (n = 229) performed between June 2005 and December 2007 were entered into a prospective study of 1-h Bx and stained for C4d by immunofluorescence. Transplants were performed against a negative T-cell CDC-XM with the exception of three cases with a positive B-cell XM. Results. All 229 1-h Bx stained negative for C4d. Fourteen pts (6%) developed AMR. None of the 14 protocol 1-h Bx stained positive for C4d in peritubular capillaries (PTC). However, all indication biopsies-that diagnosed AMR-performed at a median of 8 days after transplantation stained for C4d in PTC. Conclusions. These data show that C4d staining in 1-h Bx is, in general, not useful for the early detection of AMR when CDC-XM is negative.

Antibody Response from Whole-Cell Pertussis Vaccine Immunized Brazilian Children against Different Strains of Bordetella pertussis

Bordetella pertussis is a gram-negative bacillus that causes the highly contagious disease known as pertussis or whooping cough. Antibody response in children may vary depending on the vaccination schedule and the product used. In this study, we have analyzed the antibody response of cellular pertussis vaccinated children against B. pertussis strains and their virulence factors, such as pertussis toxin, pertactin, and filamentous hemagglutinin. After the completion of the immunization process, according to the Brazilian vaccination program, children serum samples were collected at different periods of time, and tested for the presence of specific antibodies and antigenic cross-reactivity. Results obtained show that children immunized with three doses of the Brazilian whole-cell pertussis vaccine present high levels of serum antibodies capable of recognizing the majority of the components present in vaccinal and non-vaccinal B. pertussis strains and their virulence factors for at least 2 years after the completion of the immunization procedure.

The macrophage-derived neutrophil chemotactic factor, MNCF: A lectin with TNF-alpha-like activities on neutrophils

The macro phage-derived neutrophil chemotactic factor (MNCF) is an alpha-galactoside-binding lectin, known to induce dexamethasone-insensitive neutrophil recruitment. We further characterized MNCF effects on neutrophils and showed that it shares with TNF-alpha the ability to delay apoptosis and to trigger degranulation. MNCF and TNF-alpha effects show similar kinetics and involve Src kinases and MAPKinases dependent pathways. They were, however, clearly distinguished, since the soluble TNF-receptor etanercept prevented TNF but not MNCF effects, while melibiose disaccharide inhibited MNCF but not TNF effects. Absorption of MNCF on detoxi-gel did not alter its properties, precluding an LPS contamination effect. By contrast, galectin-3 required LPS to activate neutrophils. Specific antibodies allowed to further demonstrate that MNCF and galectin-3 are two distinct molecules. Finally, MNCF- and IL-8-induced neutrophil activation differed by their kinetic and sensitivity to pertussis toxin. In conclusion, MNCF is a distinct neutrophil agonist, with pro-inflammatory activities involving its carbohydrate recognition domain. (C) 2008 Elsevier Inc. All rights reserved.

Analysis of the Ontogenetic Variation in the Venom Proteome/Peptidome of Bothrops jararaca Reveals Different Strategies to Deal with Prey

Previous studies have demonstrated that the pharmacological activities displayed by Bothrops jararaca venom undergo a significant ontogenetic shift. Variation in the venom proteome is a well-documented phenomenon; however, variation in the venom peptidome is poorly understood. We report a comparative proteomic and peptidomic analysis of venoms from newborn and adult specimens of B. jararaca and correlate it with the evaluation of important venom features. We demonstrate that newborn and adult venoms have similar hemorrhagic activities, while the adult venom has a slightly higher lethal activity in mice; however, the newborn venom is extremely more potent to kill chicks. The coagulant activity of newborn venom upon human plasma is 10 times higher than that of adult venom. These differences were clearly reflected in their different profiles of SDS-PAGE, gelatin zimography, immunostaining using specific antibodies, glycosylation pattern, and concanavalin A-binding proteins. Furthermore, we report for the first time the analysis of the peptide fraction of newborn and adult venoms by MALDI-TOF mass spectrometry and LC-MS/MS, which revealed different contents of peptides, while the bradykinin potentiating peptides (BPPs) showed rather similar profiles and were detected in the venoms showing their canonical sequences and also novel sequences corresponding to BPPs processed from their precursor protein at sites so far not described. As a result of these studies, we demonstrated that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and in animal size are associated with changes in the venom proteome in B. jararaca species.

Immune response to Bacteroides forsythus in a murine model

A murine skin abscess model was used to study the immune response to an acute infection with Bacteroides forsythus. BALB/c mice were given subcutaneous injections of either viable or heat-killed B. forsythus, while a third sham-immunized control group received phosphate-buffered saline. Weights and lesion sizes were measured. Blood was collected from the heart and specific antibodies to B. forsythus measured by an ELISA. Swabs taken from the lesions and also from pooled blood were cultured anaerobically for viable B. forsythus. Viable B. forsythus-induced lesions reached maximum size at day 7. B. forsythus cells were recovered from lesions up to day 4 although none were cultured from blood samples. Heat-killed bacteria induced much smaller lesions. Serum antibody levels increased during the 9-day study period, being significantly higher in mice injected with viable compared with heat-killed B. forsythus. Antibody levels in sham control mice were significantly lower than those seen in the other two groups. These results showed that a subcutaneous injection of viable cells of B. forsythus elicited a pronounced abscess formation and induce higher levels of specific antibodies compared with that produced by an injection of dead bacteria. This suggests that, as with other periodontopathic organisms, this mouse model can be used to study the immune response to B. forsythus.

Antibody levels to the class I and II epitopes of the M protein and myosin are related to group A streptococcal exposure in endemic populations

Rheumatic fever (RF)/rheumatic heart disease (RHD) and post-streptococcal glomerulonephritis are thought to be autoimmune diseases, and follow group A streptococcal (GAS) infection. Different GAS M types have been associated with rheumatogenicity or nephritogenicity and categorized into either of two distinct classes (I or II) based on amino acid sequences present within the repeat region ('C' repeats) of the M protein. Sera from ARF patients have previously been shown to contain elevated levels of antibodies to the class I-specific epitope and myosin with the class I-specific antibodies also being cross-reactive to myosin, suggesting a disease association. This study shows that immunoreactivity of the class I-specific peptide and myosin does not differ between controls and acute RF (ARF)/RHD in populations that are highly endemic for GAS, raising the possibility that the association is related to GAS exposure, not the presence of ARF/RHD. Peptide inhibition studies suggest that the class I epitope may be conformational and residue 10 of the peptide is critical for antibody binding. We demonstrate that correlation of antibody levels between the class I and II epitope is due to class II-specific antibodies recognizing a common epitope with class I which is contained within the sequence RDL-ASRE. Our results suggest that antibody prevalence to class I and II epitopes and myosin is associated with GAS exposure, and that antibodies to these epitopes are not an indicator of disease nor a pathogenic factor in endemic populations.

Effect of Fusobacterium nucleatum on the T and B cell responses to Porphyromonas gingivalis in a mouse model

T cell cytokine profiles and specific serum antibody levels in five groups of BALB/c mice immunized with saline alone, viable Fusobacterium nucleatum ATCC 25586, viable Porphyromonas gingivalis ATCC 33277, F. nucleatum followed by P. gingivalis and P. gingivalis followed by F nucleatum were determined. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by dual colour flow cytometry and the levels of serum anti-F. nucleatum and anti-P. gingivalis antibodies determined by an ELISA. Both Th1 and Th2 responses were demonstrated by all groups, and while there were slightly lower percentages of cytokine positive T cells in mice injected with F. nucleatum alone compared with the other groups immunized with bacteria., F nucleatum had no effect on the T cell production of cytokines induced by P gingivalis in the two groups immunized with both organisms. However, the percentages of cytokine positive CD8 cells were generally significantly higher than those of the CD4 cells. Mice immunized with F nucleatum alone had high levels of serum anti-E nucleatum antibodies with very low levels of P. gingivalis antibodies, whereas mice injected with P gingivalis alone produced anti-P. gingivalis antibodies predominantly. Although the levels of anti-E nucleatum antibodies in mice injected with E nucleatum followed by P. gingivalis were the same as in mice immunized with F nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F nucleatum produced equal levels of both anti-P. gingivalis and anti-F nucleatum antibodies, although at lower levels than the other three groups immunized with bacteria, respectively. Anti-Actinobacillus actitiomycetemcomitans, Bacteroides forsythus and Prevotella intermedia serum antibody levels were also determined and found to be negligible. In conclusion, F nucleatum immunization does not affect the splenic T cell cytokine response to P. gingivalis. However, F nucleatum immunization prior to that of P. gingivalis almost completely inhibited the production of anti-P gingivalis antibodies while P. gingivalis injection before F. nucleatum demonstrated a partial inhibitory effect by P. gingivalis on antibody production to F. nucleatum. The significance of these results with respect to human periodontal disease is difficult to determine. However, they may explain in part differing responses to P. gingivalis in different individuals who may or may not have had prior exposure to F. nucleatum. Finally, the results suggested that P. gingivalis and F. nucleatum do not induce the production of cross-reactive antibodies to other oral microorganisms.