Survival of Anaplasma marginale in Aedes albopictus cells

Bovine red blood cells infected with Anaplasma marginale were introduced into cell line cultures from the mosquito Aedes albopictus. The cultured mosquito cells phagocytized both infected and uninfected red blood cells. The parasite remained viable, as determined by calf inoculations, for 21 days.     

The repertoire of Anaplasma marginale antigens recognized by CD4(+) T-lymphocyte clones from protectively immunized cattle is diverse and includes major surface protein 2 (MSP-2) and MSP-3

Major surface proteins of Anaplasma marginale are vaccine candidates. We recently demonstrated that immunization of calves with outer membranes of the Florida strain of A. marginale resulted in protective immunity that correlated with a memory CD4(+) T-lymphocyte response specific for major surface protein 1 (MSP-1), MSP-2, and MSP-3 (W. C. Brown, V. Shkap, D. Zhu, T. C. McGuire, W. Tuo, T. F. McElwain, and G. H. Palmer, Infect. Immun. 66:5406-5413, 1998). As immunogens, these proteins have been shown to induce complete or partial protection against homologous challenge. To further define the T helper (Th) cell response to these and other A. marginale antigens and to determine conservation of Th cell epitopes among genetically distinct A. marginale strains, Th cell clones obtained prior to challenge from three immunized calves were characterized for antigen-specific responses. Nine distinct antigenic profiles were defined by 11 Th cell clones derived by stimulation with the Florida strain. Several clones responded to MSP-2, MSP-3, or both. All of these MSP-2- or MSP-3-specific clones and the majority of other clones that did not respond to MSPs recognized all bovine blood-passaged strains of A. marginale. These results demonstrate conservation of certain Th cell epitopes between MSP-2 and MSP-3 and show that Th cell epitopes in MSP-2, MSP-3, and undefined antigens are conserved among strains of A. marginale. Of seven clones that responded to the blood-passaged Virginia strain, two did not recognize antigen prepared from this strain cultured in tick cells, suggesting differences in the antigenic composition between these stages. Analysis of the cytokines expressed by the Th cells revealed that all clones expressed gamma interferon and tumor necrosis factor alpha, and most coexpressed interleukin-4. Our results provide a rationale for identifying Th cell epitopes conserved among different strains of A. marginale for inclusion in a nucleic acid or recombinant protein vaccine.     

Development of Anaplasma marginale in salivary glands of male Dermacentor andersoni

The objective of this study was to clarify the role of oestradiol in luteal function by examining its effect on the oxytocin stimulation of 15-keto-13,14-dihydro-prostaglandin F2 alpha (PGFM) concentrations in cyclic mares. In the first experiment, three groups of mares (4 per group) were given a bolus injection of 17 alpha-oestradiol (1 mg), oestradiol (1 mg) or vehicle on days 7, 9, 11, 13 and 15 of the cycle. Six hours later the mares were challenged with 10 iu oxytocin intravenously and frequent blood samples were taken from 15 min before to 15 min after for measurement of PGFM. Results showed a significant stimulatory effect of oestradiol (five times greater than controls at day 11; P < 0.05), but not of 17 alpha-oestradiol, on the oxytocin stimulation of PGFM. As a relatively large dose was given systemically in this experiment, a second experiment was performed to introduce a dose that was more physiological into the uterus. Small Silastic spheres (1 cm diameter) were impregnated with or without oestradiol at a concentration that gave a release rate similar to that of embryos at day 12 (10 ng h-1). These were inserted (one per mare) into the uterus of two groups of mares (five per group) on day 7. The mares were challenged with oxytocin on days 9, 11, 13 and 15 of the cycle and blood samples were taken as before for determination of PGFM. The results showed that oestradiol enhanced (four times greater than controls at day 13; P < 0.05) the oxytocin stimulation of PGFM concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of dexamethasone on the recrudescence of Anaplasma marginale in splenectomized calves

Dexamethasone was administered at the dose rate of 0.2 mg/kg of body weight to 11 splenectomized Anaplasma-carrier calves (groups 1 and 3) on Monday, Wednesday, and Friday for 3 weeks. Observations were made on these calves and on 7 nontreated, comparable calves (group 2) to determine the influence of treatment on carrier infections. Dexamethasone treatment was associated in every instance with an exacerbation of the Anaplasma parasitemia and a decrease in packed red cell volume. The episode of acute anaplasmosis was of short duration, resembling the primary response, except that complement-fixation response did not increase accordingly. Serum protein electrophoresis of serums from 4 calves (group 3) undergoing the drug-induced response failed to show any significant change during the 3-week treatment period, but did show a significant increase in gamma-globulin immediately after treatment.     

B-cell epitopes recognized by IgE from patients sensitive to Amb a 5

BACKGROUND: The response to allergens characterized by IgE-mediated hypersensitivity is selective. The search for the inherited contribution to atopy has among other things, focused on the linkage of sensitivity to the presence of specific alleles in the DR and DQ locus. More than 90% of the responders to Amb a 5, an allergen from ambrosia artemisifolia, are DR-2 positive. This relationship is logically linked to the T-cell epitope presentation by the HLA complex. OBJECTIVES: This study aims to investigate a possible relationship between T-cell epitopes, B-cell epitopes and the alleles of the DR and DQ loci in Amb a 5 sensitive DR-2+ and DR-2- individuals. METHODS: Inhibition of solid state Elisa assays by IgE-enriched and IgG-depleted, heated sera. The inhibition was carried out in checkerboard pattern, bidirectionally; A inhibits B and B inhibits A. RESULTS: The B-cell epitopes defined by the inhibition pattern were all found to be conformational. Three different epitope patterns (A, B, C) were recognized. The IgE and IgG complexes were found in only one responder. The DR and DQ locus alleles were all sequenced. Although all the individuals studied responding to Amb a 5 show presence of alleles such as 1501, associated with DR-2, our data indicates no correlation between the B-cell epitopes recognized and the DR and DQ locus alleles. A well known, general T-cell motif was recognized in the known sequence of Amb a 5. CONCLUSIONS: Our investigation suggests that the choice of B-cell recognition is regulated independently of a putative link between T-cell epitope recognition and the D locus.     

Short report: identification of B-cell epitopes in the serine-rich Entamoeba histolytica protein

The serine-rich Entamoeba histolytica protein (SREHP) has been shown to be a protective antigen in animal models of amebic liver abscess when delivered by either parenteral or oral routes of immunization, and antibodies to SREHP can prevent amebic liver abscess in severe combined immunodeficient mice. To identify B cell epitopes of the SREHP molecule that could serve as the basis for a peptide-based vaccine, we synthesized overlapping peptides spanning the amino acid sequence of SREHP, and looked at the reactivity of serum samples from five individuals with amebic liver abscess to the overlapping peptides. We found that most of the epitopes recognized by serum samples from patients with amebic liver abscess map to the hydrophilic dodecapeptide or octapeptide repeats of SREHP, but there was no universal epitope recognized by all five serum samples. In addition, we show that synthetic peptides that include the epitopes of SREHP recognized in the mapping study are immunogenic in animals and can generate antibodies that recognize SREHP.     

Dominance of conserved B-cell epitopes of the Plasmodium falciparum merozoite surface protein, MSP1, in blood-stage infections of naive Aotus monkeys

We have shown that conserved B epitopes were immunodominant in animals hyperimmunized with parasite-purified or recombinant merozoite surface protein MSP1 of Plasmodium falciparum. Cross-priming studies also suggested that a conserved T-helper epitope(s) is efficient in inducing the anti-MSP1 antibody response. In this study, we determined whether a similar profile of immune responses was induced during live P. falciparum infections. Naive Aotus monkeys were infected by blood-stage challenge with either one of the two dimorphic MSP1 alleles represented by the FUP and FVO parasites. Sera collected after parasite clearance were analyzed by enzyme-linked immunosorbent assays (ELISAs). Monkeys infected with parasites carrying one allelic form of MSP1 had antibodies that were equally reactive with homologous or heterologous MSP1s. This preferential recognition of conserved epitopes of MSP1 was confirmed by competitive binding ELISAs. Studies with Plasmodium yoelii and P. falciparum show that the C-terminal 19-kDa fragment of MSP1, MSP1(19), is the target of protective immunity. Thus, monkey sera were assayed for recognition with recombinant MSP1(19)s expressing variant and conserved B epitopes. Results of direct and competitive binding ELISAs showed that the anti-MSP1(19) antibodies were also directed primarily against conserved determinants. The similarities between vaccine- or infection-induced antibody responses suggest a possible reciprocal enhancement of the two populations of anti-MSP1 antibodies when a subunit MSP1 vaccine is introduced into populations living in areas where malaria is endemic. This together with previous observations that conserved determinants are important in MSP1-mediated immunity provides an optimistic outlook that a subunit MSP1 vaccine may be effective and practical for field applications in malaria-exposed populations.     

Identification of a major surface protein on Neospora caninum tachyzoites

Neospora caninum is a recently identified coccidian parasite that is closely related to Toxoplasma gondii. Molecules associated with the surface of N. caninum tachyzoites are likely to be involved in the process of adhesion and invasion of host cells. They probably also participate in the interaction of the parasite with the immune system, and they could play an important role in the pathogenesis of the parasite. To identify such surface molecules, we performed subcellular fractionation studies of isolated N. caninum tachyzoites. Employing the nonionic detergent Triton-X-114, we prepared a membrane fraction. Immunoblot analysis of this fraction using polyclonal antisera directed against tachyzoites of N. caninum and T. gondii resulted in the identification of a protein of approximately 43 kDa (Nc-p43). This molecule was present in two isolates of Neospora (Nc-1 and Liverpool) but was absent in Toxoplasma (RH-strain) tachyzoites. Further immunofluorescence and immunogold transmission electron microscopy (TEM) studies using affinity-purified anti-Nc-p43 antibodies demonstrated the presence of this molecule on the surface of N. caninum tachyzoites.     

Mapping and comparison of the B-cell epitopes recognized on the Plasmodium vivax circumsporozoite protein by immune Colombians and immunized Aotus monkeys

We present a protocol for in vitro immunization of B cells using monocyte-derived accessory cells (MoAC). MoAC are developed from human peripheral blood monocytes in culture and represent functionally competent inducers of antigen-specific immune responses. Using MoAC, we attempted to immunoselect TT-specific lymphocytes by rosetting. Adherent human MoAC were pulsed with tetanus toxoid (TT) and allowed to form clusters with autologous lymphocytes, followed by removal of non-adherent cells. After one week of culture, a specific anti-TT antibody response emerged on a low background of unspecific Ig. In comparison, cultures which had not been selected for adherent cells produced a high polyclonal background. Our results demonstrate that from peripheral blood cells, previously not a favourable source for in vitro immunization, in a majority of tests antigen-specific B cells could efficiently be immunoselected via adherence to autologous antigen-presenting cells, leading to a high-titre in vitro immunization.     

Identification and characterization of human B-cell epitopes in recombinant antigens of Leishmania aethiopica

Recombinant DNA fragments from Leishmania aethiopica that code for epitopes which react with human antibodies have been characterized by cross-hybridization studies and DNA sequence analysis. Twenty clones could be grouped into seven different groups (I-VII), probably representing seven different L. aethiopica antigens. The DNA sequences of representative clones from the seven groups have been obtained and the amino acid sequence of the respective recombinant antigens established. The recombinant antigens have been analysed by epitope scanning with patient sera, and octapeptides that contain potential B-cell epitopes have been identified in all seven recombinant antigens. These octapeptides have further been tested with additional patient sera and control sera, and three octapeptides (HAFCHEEG, YHSSVVHD and SYAPCSLK) were found to contain major epitopes recognizing specific antibodies in nine, seven and four, respectively, of the twenty sera tested. Fifteen of the twenty sera reacted with one or more of these three octapeptides.     

Induction of native protein reactive antibodies by immunization with peptides containing linear B-cell epitopes defined by anti-porcine ZP3 beta monoclonal antibodies

To identify pertinent target epitopes for contraceptive vaccine development, rabbit polyclonal antibodies were raised against four peptides synthesized from the deduced amino acid (aa) sequence of porcine zona pellucida macromolecule ZP3 beta and coupled to diphtheria toxoid (DT). Synthetic peptides consisted of: P1, 23-37 aa; P2, 164-179 aa with an additional C-terminal cysteine; P3, 246-263 aa with an extra C-terminal cysteine; and P4, 310-321 aa residues corresponding to pZP3 beta precursor protein. Selected sequences were based upon B cell epitopes identified previously by monoclonal antibodies. Immune sera reacted with their respective peptides and DT in an ELISA, and also recognized porcine SIZP and pZP3 beta both in ELISA and Western blot and zona pellucida of porcine oocytes in an indirect immunofluorescence assay. None of the four anti-peptide sera recognized pZP3 alpha in Western blot, emphasizing the specificity of these antibodies to pZP3 beta. The anti-peptide sera, individually, failed to inhibit in vitro attachment of boar sperm to antibody treated zona encased porcine oocytes. However, combinations of immune sera against peptides such as P1 + P4, P2 + P4 and P1 + P2 + P4, did significantly inhibit porcine sperm-oocyte interaction. These results identify combinations of peptides that could potentially be used in the design of an immunocontraceptive vaccine based upon synthetic peptides corresponding to pZP3 beta or its homologues in other species.     

Linear B-cell epitopes in the N-terminus of L2 of bovine papillomavirus type 4

In vivo dosimetry performed with semiconductor detectors is a reliable method for patient dose control. The purpose of this study is to evaluate the perturbations introduced in the patient's absorbed dose distribution by three types of commercially available diodes (Isorad, Sun Nuclear Corp.; model 114200, 114300 and 114400) from the same company and to present possible solutions for minimizing this side-effect.     

Recognition of B-cell epitopes of the 65 kDa HSP in Beh?et's disease

B-cell epitopes of the mycobacterial 65 kDa heat shock protein (HSP) were mapped in sera from patients with Beh?et's Disease (BD). A series of 47 overlapping synthetic peptides (15ers) derived from the sequence of the Mycobacterium tuberculosis 65 kDa HSP was used in ELISA. Significant increases in IgA and IgG antibody levels were observed with peptides 111-125, 154-172 and 311-326 in sera from BD, compared with those from controls. Homologous peptides derived from the sequence of the human mitochondrial 60 kDa HSP were then examined. Peptides 136-150 and 336-351 showed comparable results to the homologous mycobacterial peptides 111-125 and 311-326, respectively. The B-cell epitopes defined in this investigation overlap with the T-cell epitopes the authors have previously reported in BD. Inhibition studies are consistent with the view that antibodies to each of the three B-cell epitope peptides represent a small proportion of the total B-cell epitope repertoire elicited by the 65 or 60 kD HSP. Sequential antibody studies suggest that IgA and IgG antibody titres to one or all three peptides tested may increase during exacerbation of ocular disease. The functional role of these antibodies needs to be determined, but the peptides may be involved in the immunopathogenesis of BD as they can induce experimental uveitis in Lewis rats, which is a principal manifestation of BD.     

Identification and characterization of epitopes on the 120-kilodalton surface protein antigen of Rickettsia prowazekii with synthetic peptides

The 120-kDa surface protein antigens (SPAs) of typhus rickettsiae are highly immunogenic and have been shown to be responsible for the species-specific serological reactions of the typhus group rickettsiae. To study the immunochemistry of these proteins, overlapping decapeptides encompassing the whole protein were synthesized on derivatized polyethylene pins. A modified enzyme-linked immunosorbent assay was used to identify epitopes recognized by rabbit hyperimmune antisera to Rickettsia prowazekii SPA. Eight distinct epitopes were mapped by this method in three regions. Four of the epitopes, which were located in the carboxyterminus of mature processed SPA, were strongly competitively inhibited by native folded SPA but not by intact rickettsiae, suggesting that they were on the SPA surface but not exposed on the rickettsial surface. Three of these epitopes were present on both R. prowazekii and Rickettsia typhi SPAs. The immunoreactivities of five epitopes were further characterized by synthesizing modified peptides. Glycine substitution experiments determined the critical residues in the epitopes. The dependence of binding of the peptide epitopes to the polyclonal antisera was mapped to single residues. The limited number and weak reactivity of linear peptide epitopes observed with human and rabbit sera, possibly due to a lack of the methylated amino acids which are present in rickettsia-derived SPA, suggest that the present approach will not provide useful synthetic antigens for diagnosis of typhus infections.     

Passive immunization with antibodies against three distinct epitopes on Plasmodium yoelii merozoite surface protein 1 suppresses parasitemia

We have produced monoclonal antibodies against Plasmodium yoelii merozoite surface protein 1 (MSP-1) and have assessed their ability to suppress blood stage parasitemia by passive immunization. Six immunoglobulin G antibodies were characterized in detail: three (B6, D3, and F5) were effective in suppressing a lethal blood stage challenge infection, two (B10 and G3) were partially effective, and one (B4) was ineffective. MSP-1 is the precursor to a complex of polypeptides on the merozoite surface; all of the antibodies bound to this precursor and to an approximately 42-kDa fragment (MSP-142) that is derived from the C terminus of MSP-1. MSP-142 is further cleaved to an N-terminal approximately 33-kDa polypeptide (MSP-133) and a C-terminal approximately 19-kDa polypeptide (MSP-119) comprised of two epidermal growth factor (EGF)-like modules. D3 reacted with MSP-142 but not with either of the constituents MSP-133 and MSP-119, B4 recognized an epitope within the N terminus of MSP-133, and B6, B10, F5, and G3 bound to MSP-119. B10 and G3 bound to epitopes that required both C-terminal EGF-like modules for their formation, whereas B6 and F5 bound to epitopes in the first EGF-like module. These results indicate that at least three distinct epitopes on P. yoelii MSP-1 are recognized by antibodies that suppress parasitemia in vivo.     

Mtv-1 superantigen trafficks independently of major histocompatibility complex class II directly to the B-cell surface by the exocytic pathway

Presentation of the Mtv-1 superantigen (vSag1) to specific Vbeta-bearing T cells requires association with major histocompatibility complex class II molecules. The intracellular route by which vSag1 trafficks to the cell surface and the site of vSag1-class II complex assembly in antigen-presenting B lymphocytes have not been determined. Here, we show that vSag1 trafficks independently of class II to the plasma membrane by the exocytic secretory pathway. At the surface of B cells, vSag1 associates primarily with mature peptide-bound class II alphabeta dimers, which are stable in sodium dodecyl sulfate. vSag1 is unstable on the cell surface in the absence of class II, and reagents that alter the surface expression of vSag1 and the conformation of class II molecules affect vSag1 stimulation of superantigen reactive T cells.