A nano particle vector comprised of poly lactic-co-glycolic acid and monophosphoryl lipid A and recombinant Mycobacterium avium subsp paratuberculosis peptides stimulate a pro-immune profile in bovine macrophages.

AIMS: We evaluated the potential of a nanoparticle (NP) delivery system to improve methods of delivery of candidate peptide-based vaccines for Paratuberculosis in cattle. METHODS AND RESULTS: Peptides derived from Mycobacterium avium subsp. paratuberculosis (Map), and the pro-inflammatory monophosphoryl lipid A (MPLA) were incorporated in polymeric NPs based on poly (d,l-lactide-co-glycolide) (PLGA). The PLGA/MPLA NPs carriers were incubated with macrophages to examine their effects on survival and function. PLGA/MPLA NPs, with and without Map antigens, are efficiently phagocytized by macrophages with no evidence of toxicity. PLGA/MPLA NP formulations did not alter the level of expression of MHC I or II molecules. Expression of TNFα and IL12p40 was increased in Map-loaded NPs. T-cell proliferation studies using a model peptide from Anaplasma marginale demonstrated that a CD4 T-cell recall response could be elicited with macrophages pulsed with the peptide encapsulated in the PLGA/MPLA NP. CONCLUSIONS: These findings indicate PLGA/MPLA NPs can be used as a vehicle for delivery and testing of candidate peptide-based vaccines. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will assist on more in depth studies on PLGA NP delivery systems that may lead to the development of a peptide-based vaccine for cattle.

The bovine spleen: interactions among splenic cell populations in the innate immunologic control of hemoparasitic infections.

Over the past several years, innate immunity has been recognized as having an important role as a front-line defense mechanism and as an integral part of the adaptive immune response. Innate immunity in cattle exposed to hemoparasites is spleen-dependent and age-related. In this review, we discuss general aspects of innate immunity and the cells involved in this aspect of the response to infection. We also provide examples of specific splenic regulatory and effector mechanisms involved in the response to Babesia bovis, an important tick-borne hemoparasitic disease of cattle. Evidence for the regulatory and effector role of bovine splenic monocytes and DC both in directing a type-1 response through interaction with splenic NK cells and γδT-cells will be presented.

Bovine NK cells acquire cytotoxic activity and produce IFN-gamma after stimulation by Mycobacterium bovis BCG- or Babesia bovis-exposed splenic dendritic cells.

Early interactions of innate immune cell populations, such as dendritic cells (DC) and natural killer (NK) cells, can affect the ability of the acquired immune response to control infection of intracellular microorganisms. In this study, we investigated the activation of bovine NK cells by CD13(+) splenic DC stimulated with either Mycobacterium bovis BCG or Babesia bovis merozoites. Splenic DC were used either immediately after selection (cytokine(-)) or after exposure to GM-CSF, IL-4 and Flt3L for 72 h (cytokine(+)). Phenotypic analyses showed up-regulation of MHCII, CD80 and CD86 on cytokine(+) DC when compared to cytokine(-) DC. Purified NK cells (CD335(+)CD3(-)CD2(+/-)CD8alpha(+/-)) were co-cultured with microbial-exposed cytokine(-) DC or cytokine(+) DC in either transwell or cell-to-cell format and NK cell IFN-gamma production and cytotoxicity were assessed. NK cell IFN-gamma production was dependent on cell-to-cell contact. Microbial-stimulated cytokine(+) DC induced significantly more IFN-gamma production from NK cells than cytokine(-) cells. In contrast, cytotoxicity and perforin up-regulation were more pronounced in NK cells cultured with cytokine(-) DC than cytokine(+) DC. Therefore, activation of bovine NK cells by microbial-stimulated CD13(+) splenic DC is influenced by the maturation state of the DC suggesting different roles for the splenic DC during disease-induced maturation.

Haemonchus contortus intestine: a prominent source of mucosal antigens.

We sought to identify antigens from Haemonchus contortus, an abomasal nematode of small ruminants, that stimulate local (abomasal lymph node, ALN) CD4+ T lymphocyte responses during a primary infection. Results led to a focus on antigens from the parasite intestine. The H. contortus intestine proved to be a major source of antigens that stimulated ALN CD4+, CD25+ T lymphocyte responses during infections in lambs. When stimulated by intestinal antigens, ALN lymphocytes from these lambs expressed IL-4 and IL-13 transcripts, and, more variably, IFN-gamma. An immunoaffinity-purified fraction, enriched for H. contortus apical intestinal membrane proteins, stimulated similar ALN responses. On further fractionation, antigens from six size classes (ranging from 30 to 200 kDa) also stimulated proliferation of ALN lymphocytes. Mass spectrometry analysis of these size classes identified several known apical intestinal membrane proteins from H. contortus. The results show that H. contortus intestinal antigens warrant investigation in strategies to induce mucosal immunity against this parasite. The specific proteins identified have value for this purpose. The results are in contrast with the now generalized idea that H. contortus intestinal antigens are 'hidden' from the host immune system, and this issue is discussed. The approach also has potential application to other gastrointestinal nematode parasites.

Differential response of splenic monocytes and DC from cattle to microbial stimulation with Mycobacterium bovis BCG and Babesia bovis merozoites.

Both bovine peripheral blood monocyte-derived dendritic cells (DC) and myeloid DC from afferent lymph have been described, but resident DC from other bovine tissues have not been fully characterized. The spleen as a secondary lymphoid organ is central to the innate and acquired immune response to various diseases particularly hemoprotozoan infections like babesiosis. Therefore, we developed methods to demonstrate the presence of myeloid DC from the spleen of cattle and have partially characterized a DC population as well as another myeloid cell population with monocyte characteristics. The phenotypic profile of each population was CD13+CD172a+/-CD14-CD11a-CD11b+/-CD11c+ and CD172a+CD13+/-CD14+CD11a-CD11b+/-CD11c+, respectively. The CD13+ population was found exclusively in the spleen whereas the CD172a+ population was present at the same percentage in the spleen and peripheral blood. CD13+ cells developed a typical veiled appearance when in culture for 96 h. The two cell populations differed in their ability to produce nitric oxide and had a different pattern of cytokine mRNA when stimulated with Mycobacterium bovis BCG or Babesia bovis merozoites. The data demonstrate the presence of a myeloid splenic DC with attributes consistent with an immature status.

Prospects for recombinant vaccines against Babesia bovis and related parasites.

Babesial parasites infect cattle in tropical and temperate regions of the world and cause significant morbidity and mortality. Discovery of protective antigens that could be used in a killed vaccine has been slow and to date there are few promising vaccine candidates for cattle Babesia. This review describes mechanisms of protective innate and adaptive immune responses to babesial parasites and different strategies to identify potentially protective protein antigens of B. bovis, B. bigemina, and B. divergens. Successful parasites often cause persistent infection, and this paper also discusses how B. bovis evades and regulates the immune response to promote survival of parasite and host. Development of successful non-living recombinant vaccines will depend on increased understanding of protective immune mechanisms and availability of parasite genomes.

Development of detection methods for ruminant interleukin (IL)-4.

Recombinant bovine IL-4 (rbo IL-4) was transiently expressed in COS-7 cells. Mice were immunised with a plasmid encoding rbo IL-4 and boosted with rbo IL-4. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-4 in an ELISA and these cloned hybridomas were termed CC311, CC312, CC313 and CC314. A pair of mAb (CC313 and CC314) was identified that together could be used to detect both recombinant and native bovine IL-4 by ELISA and a luminometric detection method was applied to the ELISA. Using this method native bovine IL-4 was detected in supernatants of PBMC stimulated with mitogens. In addition, high level secretion of IL-4 by Fasciola hepatica specific Th2 clones, but not by a Babesia bovis specific Th1 clone, was confirmed. The ELISA was also able to detect recombinant ovine IL-4. The pair of mAb used for ELISA could also be used for the detection of IL-4 spot forming cells by ELISPOT. In addition intracytoplasmic expression of IL-4 could be detected. The ability to detect ruminant IL-4 by three methods: ELISA, ELISPOT and by flow cytometric analysis of intracytoplasmic expression will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

Low molecular weight proteins of Cowdria ruminantium (Welgevonden isolate) induce bovine CD4+-enriched T-cells to proliferate and produce interferon-gamma.

An important objective in vaccination strategies is to activate lymphocytes with particular effector functions. Cellular immunity and the type I cytokine IFN-gamma have been implicated in protective immunity to heartwater. Furthermore, low molecular weight proteins of Cowdria ruminantium have been shown to induce peripheral blood mononuclear cells to proliferate. To determine which lymphocyte subset responds when stimulated with fractionated C. ruminantium proteins, specific short-term lymphocyte cultures were established from cattle immunized with the Welgevonden isolate. Four cattle were immunized, two by infection and treatment and two with inactivated organisms. Cell surface phenotypic analysis of the cultures indicated that CD4+ lymphocytes were enriched over time. This coincided with increased antigen-specific proliferation and IFN-gamma production. Proteins of molecular weights 13-18kDa induced the CD4+-enriched T-cell cultures, derived from each of the animals, to proliferate and produce IFN-gamma. Although the two groups of cattle were immunized differently, their lymphocytes responded similarly. These results extend previous findings by identifying the responder cells as being predominantly IFN-gamma producing CD4+ lymphocytes. This cytokine has been implicated in immunity to the parasite. The low molecular weight proteins that induced CD4+ lymphocytes to proliferate and produce IFN-gamma are therefore likely to be important in protection against heartwater and may have a role in vaccine development.

Induction of interleukin-6 and interleukin-12 in bovine B lymphocytes, monocytes, and macrophages by a CpG oligodeoxynucleotide (ODN 2059) containing the GTCGTT motif.

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) that contain unmethylated CpG dinucleotides flanked by certain bases (CpG ODN) have been shown to activate murine and human B cells and to induce proinflammatory cytokines by monocytes/macrophages and dendritic cells (DC). However, the CpG ODN sequences optimal for mice and humans are different. In the current study, the effects of CpG ODN, which were defined to stimulate strong responses in either mouse or human leukocytes, were compared for stimulation of bovine B lymphocyte proliferation and macrophage cytokine mRNA expression. The optimal CpG ODN was then tested for induction of cytokines in peripheral blood mononuclear cells (PBMC) and purified B lymphocytes, monocytes, and macrophages. At a high ODN concentration (40 microM), all but two CpG ODN tested stimulated B cell proliferation, which was dependent on unmethylated CpG motifs. CpG ODN 2059 containing the GTCGTT motif shown to activate human leukocytes also promoted the highest level of bovine B cell proliferation at a lower concentration (10 microM) when compared with CpG ODN containing AACGTT or GACGTT motifs active for murine leukocytes. Furthermore, ODN 2059 induced interleukin-6 (IL-6) production by B lymphocytes and IL-6 and IL-12 production by PBMC, monocytes, and macrophages. In contrast, IL-1beta and tumor necrosis factor-alpha (TNF-alpha) production was either very low or undetectable. Consistent with increased IL-12 production, ODN 2059 also stimulated interferon-gamma (IFN-gamma) production by PBMC. Importantly, the levels of cytokines induced by ODN 2059 were comparable to those generated in response to Escherichia coli DNA. The weak TNF-alpha response combined with the vigorous IL-6 and IL-12 response to ODN 2059 indicate the potential use of this CpG ODN as an adjuvant to enhance both antibody-mediated and IFN-gamma-mediated macrophage activation, which are important for protection against disease caused by intracellular pathogens of cattle.

Molecular approaches to elucidating innate and acquired immune responses to Babesia bovis, a protozoan parasite that causes persistent infection.

For many vector-transmitted protozoal parasites, immunological control of acute infection leads to a state of persistent infection during which parasitemias may cycle unnoticed in infected but otherwise clinically healthy animals. Achieving persistent infection is a strategy that favors parasitism, since both host and, therefore, parasite survive, and endemically infected animal populations provide a reservoir of parasites continually available for subsequent transmission. Examples of the major economically important protozoan pathogens that cause persistent infection in mammals include the related Theileria and Babesia parasites as well as Trypanosoma species. Control of acute infection and maintenance of clinical immunity against subsequent infection are determined by the interplay of innate and acquired immune responses. This review will focus on approaches taken to gain an understanding of the molecular basis for innate and acquired immunity against the hemoprotozoan parasite of cattle, Babesia bovis. Knowledge of mechanisms used by the parasite to survive within infected cattle from acute to persistent infection combined with definition of the correlates of protective immunity in cattle should be applicable to designing effective vaccines.

A novel 20-kilodalton protein conserved in Babesia bovis and B. bigemina stimulates memory CD4(+) T lymphocyte responses in B. bovis-immune cattle.

Acquired immunity against the hemoprotozoan parasite Babesia bovis is believed to depend on activation of antigen-specific CD4(+) T lymphocytes and IFN-gamma production. A strategy was employed to identify potentially protective antigens from B. bovis based on memory CD4(+) T lymphocyte recognition of fractionated merozoite proteins. Fractions of merozoites separated by continuous flow electrophoresis (CFE) that contained proteins of approximately 20 kDa were shown previously to stimulate memory CD4(+) lymphocyte responses in B. bovis-immune cattle with different MHC class II haplotypes. Expression library screening with rabbit antiserum raised against an immunostimulatory 20-kDa CFE fraction identified a 20-kDa protein (Bbo20) that contains a B lymphocyte epitope conserved in geographically distant B. bovis strains. An homologous 20-kDa protein that has 86.4% identity with Bbo20 and contains the conserved B cell epitope was identified in B. bigemina (Bbg20). Southern blot analysis indicated that both Babesia proteins are encoded by a single gene. Antibody against recombinant Bbo20 protein identified the antigen in CFE fractions shown previously to stimulate memory T lymphocyte responses in immune cattle. To verify Bbo20 as an immunostimulatory T lymphocyte antigen, CD4(+) T cell lines were propagated from B. bovis-immune cattle with merozoite antigen and shown to proliferate significantly against recombinant Bbo20 protein. Furthermore, Bbo20-specific CD4(+) T cell clones proliferated in response to several B. bovis strains and produced IFN-gamma. BLAST analysis revealed significant similarity of the Bbo20 and Bbg20 amino acid sequences with the hsp20/alpha-crystallin family.

CD4(+) T lymphocytes from calves immunized with Anaplasma marginale major surface protein 1 (MSP1), a heteromeric complex of MSP1a and MSP1b, preferentially recognize the MSP1a carboxyl terminus that is conserved among strains.

Native major surface protein 1 (MSP1) of the ehrlichial pathogen Anaplasma marginale induces protective immunity in calves challenged with homologous and heterologous strains. MSP1 is a heteromeric complex of a single MSP1a protein covalently associated with MSP1b polypeptides, of which at least two (designated MSP1F1 and MSP1F3) in the Florida strain are expressed. Immunization with recombinant MSP1a and MSP1b alone or in combination fails to provide protection. The protective immunity in calves immunized with native MSP1 is associated with the development of opsonizing and neutralizing antibodies, but CD4(+) T-lymphocyte responses have not been evaluated. CD4(+) T lymphocytes participate in protective immunity to ehrlichial pathogens through production of gamma interferon (IFN-gamma), which promotes switching to high-affinity immunoglobulin G (IgG) and activation of phagocytic cells to produce nitric oxide. Thus, an effective vaccine for A. marginale and related organisms should contain both T- and B-lymphocyte epitopes that induce a strong memory response that can be recalled upon challenge with homologous and heterologous strains. This study was designed to determine the relative contributions of MSP1a and MSP1b proteins, which contain both variant and conserved amino acid sequences, in stimulating memory CD4(+) T-lymphocyte responses in calves immunized with native MSP1. Peripheral blood mononuclear cells and CD4(+) T-cell lines from MSP1-immunized calves proliferated vigorously in response to the immunizing strain (Florida) and heterologous strains of A. marginale. The conserved MSP1-specific response was preferentially directed to the carboxyl-terminal region of MSP1a, which stimulated high levels of IFN-gamma production by CD4(+) T cells. In contrast, there was either weak or no recognition of MSP1b proteins. Paradoxically, all calves developed high titers of IgG antibodies to both MSP1a and MSP1b polypeptides. These findings suggest that in calves immunized with MSP1 heteromeric complex, MSP1a-specific T lymphocytes may provide help to MSP1b-specific B lymphocytes. The data provide a basis for determining whether selected MSP1a CD4(+) T-lymphocyte epitopes and selected MSP1a and MSP1b B-lymphocyte epitopes presented on the same molecule can stimulate a protective immune response.

Immunostimulatory CpG-modified plasmid DNA enhances IL-12, TNF-alpha, and NO production by bovine macrophages.

The immunogenicity of DNA vaccines is partially attributable to the adjuvant properties of bacterial plasmid DNA (pDNA) for B lymphocytes and professional antigen-presenting cells. In mice, modification of immunostimulatory sequences (ISSs), including CpG motifs, in pDNA vectors or oligodeoxynucleotides can increase or decrease their adjuvant properties. ISSs that stimulate optimal responses reportedly differ for murine and human leukocytes. We have previously characterized the mitogenic properties of oligodeoxynucleotides containing one AACGTT motif for bovine B lymphocytes. We now define cytokine responses by macrophages stimulated with pDNA engineered to contain an ISS comprising two AACGTT motifs. Macrophages activated with CpG-modified pDNA secreted significantly more interleukin-12, tumor necrosis factor-alpha, and nitric oxide than macrophages stimulated with unmodified pDNA or modified pDNA that contained nucleotides scrambled to remove CpG motifs. Engineered CpG-pDNA or CpG-oligodeoxynucleotides should be useful as vaccines or adjuvants to promote the enhanced type 1 responses important for protection against intracellular pathogens.

A unique phospholipid organization in bovine erythrocyte membranes.

Ruminant erythrocytes are remarkable for their choline-phospholipid anomalies; namely, low or absent phosphatidylcholine (PC) along with high sphingomyelin levels. Here, we report another anomaly in bovine erythrocytes that affects aminophospholipids: phosphatidylethanolamine (PE) shows an extreme asymmetry, with only 2% of the total present in the outer leaflet. Furthermore, we found that phospholipase A(2), an enzyme located on the external surface of the erythrocytes, shows higher activity against PC than against PE. In addition, we observed that acylation of PE is by far the most important biosynthetic event in this system. We propose that deacylation of PE and PC by phospholipase A(2) to generate lysocompounds, followed by selective reacylation of lyso-PE in the inner leaflet, can account for the compositional and architectural peculiarities of bovine erythrocyte membranes.

Long-term in vivo depletion of functional CD4+ T lymphocytes from calves requires both thymectomy and anti-CD4 monoclonal antibody treatment.

In vivo depletion of lymphocyte subsets is a direct approach used for dissection of the mechanisms of protective immunity. Long-term in vivo depletion of bovine T lymphocyte subpopulations with monoclonal antibody (mAb) treatment alone has been difficult to achieve. The objective of this study was to determine whether both thymectomy and anti-CD4 mAb treatment would optimize long-term in vivo depletion of functional bovine CD4+ T lymphocytes. Calves were thymectomized and treated with high doses of anti-CD4 mAb (approximately 5 mg/kg) over 4 days followed by subsequent lower doses (approximately 0.3 mg/kg) administered twice weekly for an additional 7 weeks. Depletion of CD4+ T lymphocytes from blood, spleen and peripheral lymph nodes was significantly improved in thymectomized calves compared to thymus-intact anti-CD4 mAb-treated calves. Significant differences in percentages of CD4+ T lymphocytes between thymectomized and thymus-intact calves were sustained for the duration of the 8-week study. Depletion of CD4+ T lymphocytes from thymectomized calves resulted in complete abrogation of lymphoproliferative responses to ovalbumin. In addition, thymectomized calves treated with anti-CD4 mAb had significantly reduced immunoglobulin G1 and no detectable immunoglobulin G2 ovalbumin-specific antibody responses compared to thymus-intact anti-CD4 mAb-treated calves. The results of this study demonstrate that both thymectomy and treatment with anti-CD4 mAb are required for long-term in vivo depletion of functional bovine CD4+ T lymphocytes.

DNA from protozoan parasites Babesia bovis, Trypanosoma cruzi, and T. brucei is mitogenic for B lymphocytes and stimulates macrophage expression of interleukin-12, tumor necrosis factor alpha, and nitric oxide.

The activation of innate immune responses by genomic DNA from bacteria and several nonvertebrate organisms represents a novel mechanism of pathogen recognition. We recently demonstrated the CpG-dependent mitogenic activity of DNA from the protozoan parasite Babesia bovis for bovine B lymphocytes (W. C. Brown, D. M. Estes, S. E. Chantler, K. A. Kegerreis, and C. E. Suarez, Infect. Immun. 66:5423-5432, 1998). However, activation of macrophages by DNA from protozoan parasites has not been demonstrated. The present study was therefore conducted to determine whether DNA from the protozan parasites B. bovis, Trypanosoma cruzi, and T. brucei activates macrophages to secrete inflammatory mediators associated with protective immunity. DNA from Escherichia coli and all three parasites stimulated B-lymphocyte proliferation and increased macrophage production of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO). Regulation of IL-12 and NO production occurred at the level of transcription. The amounts of IL-12, TNF-alpha, and NO induced by E. coli and protozoal DNA were strongly correlated (r2 > 0.9) with the frequency of CG dinucleotides in the genome, and immunostimulation by DNA occurred in the order E. coli > or = T. cruzi > T. brucei > B. bovis. Induction of inflammatory mediators by E. coli, T. brucei, and B. bovis DNA was dependent on the presence of unmethylated CpG dinucleotides. However, at high concentrations, E. coli and T. cruzi DNA-mediated macrophage activation was not inhibited following methylation. The recognition of protozoal DNA by B lymphocytes and macrophages may provide an important innate defense mechanism to control parasite replication and promote persistent infection.

Highly conserved regions of the immunodominant major surface protein 2 of the genogroup II ehrlichial pathogen Anaplasma marginale are rich in naturally derived CD4+ T lymphocyte epitopes that elicit strong recall responses.

Genogroup II ehrlichia, including the agent of human granulocytic ehrlichiosis, Ehrlichia phagocytophila, and the bovine pathogen Anaplasma marginale, express a markedly immunodominant outer membrane protein designated major surface protein 2 (MSP2). MSP2 is encoded by a multigene family, resulting in the expression of variant B cell epitopes. MSP2 variants are sequentially expressed in the repeated cycles of rickettsemia that characterize persistent A. marginale infection and control of each rickettsemic cycle is associated with development of a variant-specific IgG response. Importantly, these persistent rickettsemic cycles are controlled at levels 100-1000 times lower than those responsible for clinical disease during acute infection. Control of rickettsemia during persistence could result from an anamnestic Th lymphocyte response to conserved regions of MSP2 that enhances the primary Ab response against newly emergent variants. Comparison of MSP2 variants reveals conserved N and C termini flanking the central, surface-exposed hypervariable region that represents the variant B lymphocyte epitopes. We demonstrate MSP2-specific CD4(+) T lymphocyte recognition of epitopes common to several strains of A. marginale and the related pathogen A. ovis. Furthermore, T lymphocyte lines from three individuals identified six to nine overlapping peptides representing a minimum of four to seven dominant or subdominant epitopes in these conserved N and C termini. Immunodominant peptides induced high levels of IFN-gamma, a cytokine associated with protection against ehrlichia and needed for rapid generation of variant-specific IgG2. The presented data support the potential importance of a strong Th lymphocyte response to invariant MSP2 epitopes in controlling rickettsemia during persistent infection to subclinical levels.

Identification of fetal liver tyrosine kinase 3 (flt3) ligand domain required for receptor binding and function using naturally occurring ligand isoforms.

We used a comparative approach to identify the fetal liver tyrosine kinase 3 (flt3) ligand structure required for binding and function. Two conserved bovine flt3 ligand isoforms, which differ in a defined region within the extracellular domain, were identified and shown to be uniformly transcribed in individuals with diverse MHC haplotypes. Notably, at the amino acid level, the extracellular domain of the bovine flt3 ligand isoform 1 is 81 and 72% identical with the extracellular domains of the human and murine flt3 ligands, respectively, whereas isoform-2 has a deletion within this domain. Bovine flt3 ligand isoform 1, but not 2, bound the human flt3 receptor and stimulated murine pro B cells transfected with the murine flt3 receptor. This retention of binding and function allowed definition of key residues by identifying sequences conserved among species. We have shown that a highly conserved, 18 aa sequence within the flt3 ligand extracellular domain is required for flt3 receptor binding and function. However, a peptide representing this sequence is insufficient for receptor binding as demonstrated by its failure to inhibit the bovine flt3 ligand isoform 1 binding to the human flt3 receptor. The requirement for flanking structure was confirmed by testing bovine flt3 ligand isoform 1 constructs truncated at specific residues outside the 18 aa sequence. Overall, the flt3 ligand structure required for function is markedly similar to that of the related hemopoietic growth factors, CSF-1 and steel factor. This definition of the required flt3 ligand structure will facilitate development of agonists to enhance dendritic cell recruitment for vaccines and immunotherapy.

Characterization of allelic variation in the Babesia bovis merozoite surface antigen 1 (MSA-1) locus and identification of a cross-reactive inhibition-sensitive MSA-1 epitope.

The Babesia bovis merozoite surface antigen 1 (MSA-1), a member of the variable merozoite surface antigen (VMSA) family, is an immunodominant glycoprotein which elicits antibodies that inhibit erythrocyte invasion. While antigenic polymorphism is a general feature of vmsa genes, the molecular basis and extent of msa-1 sequence polymorphism have not been well characterized. In this study we defined the msa-1 locus in the biologically cloned Mexico Mo7 strain of B. bovis and identified the sequence differences between MSA-1 antigenically dissimilar strains. We then determined whether sequences conserved between distinct msa-1 alleles would induce cross-reactive CD4(+) T lymphocytes or inhibitory antibodies. The msa-1 locus in Mo7 contains a single msa-1 gene flanked by transcribed genes with no sequence homology to members of the VMSA gene family. Argentina B. bovis strains R1A and S2P have msa-1 genes with amino acid sequences that are 98.8% identical to each other, and antibodies against S2P MSA-1 cross-react with native R1A MSA-1. In contrast, identity between the Argentina and Mexico Mo7 msa-1 alleles is only 52%, with no continuous stretch of identity longer than 16 amino acids. Despite limited sequence conservation, antibodies against R1A MSA-1 were able to inhibit invasion of erythrocytes by Mo7 merozoites. The results indicate that inhibition-sensitive epitopes are conserved despite significant sequence divergence between Mexico and Argentina strain alleles and support a conserved functional role for polymorphic MSA-1 in erythrocyte invasion.