Stephen A. Hines

DNA Sequence and Comparison of Virulence Plasmids from Rhodococcus equi ATCC 33701 and 103

ABSTRACT The virulence plasmids of the equine virulent strainsRhodococcus equi ATCC 33701 and 103 were sequenced, and their genetic structure was analyzed. p33701 was 80,610 bp in length, and p103 was 1 bp shorter; their sequences were virtually identical. The plasmids contained 64 open reading frames (ORFs), 22 of which were homologous with genes of known function and 3 of which were homologous with putative genes of unknown function in other species. Putative functions were assigned to five ORFs based on protein family characteristics. The most striking feature of the virulence plasmids was the presence of a 27,536-bp pathogenicity island containing seven virulence-associated protein (vap) genes, includingvapA. These vap genes have extensive homology to vapA, which encodes a thermoregulated and surface-expressed protein. The pathogenicity island contained a LysR family transcriptional regulator and a two-component response regulator upstream of six of the vap genes. The vap genes were present as a cluster of three (vapA, vapC, and vapD), as a pair (vapE andvapF), or individually (vapG;vapH). A region of extensive direct repeats of unknown function, possibly associated with thermoregulation, was present immediately upstream of the clustered and the paired genes but not the individual vap genes. There was extensive homology among the C-terminal halves of all vap genes but not generally among the N-terminal halves. The remainder of the plasmid consisted of a large region which appears to be associated with conjugation functions and a large region which appears to be associated with replication and partitioning functions.

Characterization of the gene encoding a 60-kilodalton Babesia bovis merozoite protein with conserved and surface exposed epitopes.

A clone expressing a surface exposed, conserved epitope of a 60-kDa merozoite polypeptide was identified in a cDNA library constructed from a cloned Mexico strain of Babesia bovis. Sequencing of the 1.9-kb insert (pBv60) revealed an open reading frame encoding a 65-kDa polypeptide with a signal peptide and a tandemly repeated region. Monoclonal antibody 23/56.156, which binds a surface exposed epitope on the native polypeptide, specifically immunoprecipitated [35S]methionine-labeled polypeptides ranging from 60-30 kDa from pBv60 directed transcription and translation. Antibodies raised in rabbits against recombinant polypeptide reacted with the live merozoite surface in a polar immunofluorescence pattern, immunoprecipitated the native 60-kDa polypeptide, and were used to deplete the polypeptide by adsorption from a preparation of native [35S]methionine-labeled merozoite antigen. Restriction enzyme analysis indicated a single gene copy and the absence of introns. Hybridization demonstrated the presence of the gene in Mexico, Australia L, and Texas strains of B. bovis, but not in Babesia bigemina. A slightly different hybridization pattern was present in uncloned Australia L B. bovis, indicating sequence diversity in the Bv60 gene among isolates. Cloning and structural analysis of pBv60 provides a source of defined antigen for determining the role of conserved merozoite surface epitopes in protective immunity against babesiosis.

Identification of Pulmonary T-Lymphocyte and Serum Antibody Isotype Responses Associated with Protection against Rhodococcus equi

ABSTRACT Rhodococcus equi infects and causes pneumonia in foals between 2 and 4 months of age but does not induce disease in immunocompetent adults, which are immune and remain clinically normal upon challenge. Understanding the protective response against R. equi in adult horses is important in the development of vaccine strategies, since those mechanisms likely reflect the protective phenotype that an effective vaccine would generate in the foal. Twelve adult horses were challenged with virulent R. equi and shown to be protected against clinical disease. Stimulation of cells obtained from bronchoalveolar lavage fluid with either R. equi or the vaccine candidate protein VapA resulted in significant proliferation and a significant increase in the level of gamma interferon (IFN-γ) expression by day 7 postchallenge. The levels of interleukin-4 expression were also increased at day 7 postchallenge; however, this increase was not antigen specific. Anamnestic increases in the levels of binding to R. equi and VapA of all immunoglobulin G (IgG) antibody isotypes [IgGa, IgGb, IgG(T)] examined were detected postchallenge. The levels of R. equi- and VapA-specific IgGa and IgGb antibodies, the IgG isotypes that preferentially opsonize and fix complement in horses, were dramatically enhanced postchallenge. The antigen-specific proliferation of bronchoalveolar lavage fluid cells, the levels of IFN-γ expression by these cells, and the anamnestic increases in the levels of opsonizing IgG isotypes are consistent with stimulation of a memory response in immune adult horses and represent correlates for vaccine development in foals.

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

ABSTRACT 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 ofmsa-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. Themsa-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.

Clearance of Virulent but Not Avirulent Rhodococcus equi from the Lungs of Adult Horses Is Associated with Intracytoplasmic Gamma Interferon Production by CD4+ and CD8+ T Lymphocytes

ABSTRACT Rhodococcus equi is a gram-positive bacterium that infects alveolar macrophages and causes rhodococcal pneumonia in horses and humans. The virulence plasmid of R. equi appears to be required for both pathogenicity in the horse and the induction of protective immunity. An understanding of the mechanisms by which virulent R. equi circumvents protective host responses and by which bacteria are ultimately cleared is important for development of an effective vaccine. Six adult horses were challenged with either virulent R. equi or an avirulent, plasmid-cured derivative. By using a flow cytometric method for intracytoplasmic detection of gamma interferon (IFN-γ) in equine bronchoalveolar lavage fluid (BALF) cells, clearance of the virulent strain was shown to be associated with increased numbers of pulmonary CD4+ and CD8+ T lymphocytes producing IFN-γ. There was no change in IFN-γ-positive cells in peripheral blood, suggesting that a type 1 recall response at the site of challenge was protective. The plasmid-cured strain of R. equi was cleared in horses without a significant increase in IFN-γ-producing T lymphocytes in BALF. In contrast to these data, a previous report in foals suggested an immunomodulating role for R. equi virulence plasmid-encoded products in downregulating IFN-γ expression by equine CD4+ T lymphocytes. Intracytoplasmic detection of IFN-γ provides a method to better determine whether modulation of macrophage-activating cytokines by virulent strains occurs uniquely in neonates and contributes to their susceptibility to rhodococcal pneumonia.

The Babesia bovis Merozoite Surface Antigen 2 Locus Contains Four Tandemly Arranged and Expressed Genes Encoding Immunologically Distinct Proteins

ABSTRACT Members of the variable merozoite surface antigen (vmsa) gene family of Babesia bovis encode membrane proteins involved in erythrocyte invasion. In this study, we have identified and sequenced the complete 8.3-kb genomic locus containing msa-2, a member of the vmsa family, in the biologically cloned Mexico Mo7 strain. Four tandemly arranged copies of msa-2-related genes were found in the locus. The four genes, designated msa-2a1 (which corresponds to the originally described msa-2 gene), msa-2a2, msa-2b, and msa-2c, were shown to be transcribed and expressed and encode proteins with open reading frames ranging in size from 266 (MSA-2c) to 317 (MSA-2a1) amino acids. MSA-2a1 and -2a2 are the most closely related of the four proteins (90% identity), differing by (i) the number of 24-amino-acid repeats that comprise a surface-exposed B-cell epitope and (ii) the presence of a 32-amino-acid area of recombination between MSA-2a2 and -2b. In contrast, msa-2c is most closely related to the previously described babr 0.8 gene in Australia strains of B. bovis. Comparison of MSA-2 proteins in the Argentina R1A strain of B. bovis with the Mexico Mo7 clone revealed a relatively high degree of conservation (83.6, 69.4, 79.1, and 88.7% amino acid identity for MSA-2a1, -2a2, -2b, and -2c, respectively), in contrast to the extensive MSA-1 sequence variation (52% identity) between the same two strains. Postinfection bovine immune serum contains antibodies that bound to each of the recombinant MSA-2 proteins. Blocking assays demonstrated the presence of unique B-cell epitopes in MSA-2a1, -2b, and -2c. The results support the evolution of the msa-2 locus through at least two gene duplications, with selection for multiple related but antigenically distinct merozoite surface proteins.

Immunity to Rhodococcus equi.

Rhodococcal pneumonia is an important, life threatening disease of foals and immunosuppressed humans. Increased knowledge of the mechanisms of protective immunity are required in order to develop an effective immunoprophylaxis strategy for horses and immunotherapeutic regiments for people. Both humoral and cellular components of the immune system may be involved in immune clearance of R. equi. The susceptibility of foals less than 4-6 months of age is postulated to reflect waning maternal antibody, and passive transfer of hyperimmune plasma can provide protection on endemic farms. However, effective clearance is likely to require appropriate cellular responses, including the secretion of cytokines. In murine models, both CD4+ and CD8+ T lymphocytes can reduce bacterial counts in the lung. CD4+ cells appear to be both required and sufficient, and IFN-gamma is a primary mediator. Clearance appears to be a type 1 immune response while type 2 responses may lead to a failure to clear and lesion development. It remains to be determined how the cellular immunity experiments reported in mice relate to horses and humans. Likewise, the role of specific R. equi antigens in protective immunity has not been determined.

Analysis of anamnestic immune responses in adult horses and priming in neonates induced by a DNA vaccine expressing the vapA gene of Rhodococcus equi

Rhodococcus equi remains one of the most important pathogens of early life in horses, yet conventional vaccines to prevent rhodococcal pneumonia have not been successful. DNA vaccination offers an alternative to conventional vaccines with specific advantages for immunization of neonates. We developed a DNA vaccine expressing the vapA gene (pVR1055vapA) that induced an anamnestic response characterized by virulence associated protein A (VapA)-specific IgG antibodies in sera and bronchoalveolar lavage fluid (BALF) as well as VapA-specific proliferation of pulmonary lymphocytes when tested in adult ponies. In contrast, none of the adults receiving the control plasmid responded. To determine if pVR1055vapA induced VapA-specific responses in the foal, the targeted age group for vaccination against R. equi, 10 naïve foals were randomly assigned at birth to two groups of five. At 8-15 days of age (day 1), foals were vaccinated by intranasal and intradermal (i.d.) routes with either pVR1055vapA or the negative control pVR1055vapA_rev. All foals were DNA boosted at day 14 and protein boosted at day 30 with either recombinant VapA or recombinant CAT (control group). Prior to the protein boost, neither group developed VapA-specific immune responses. However, at day 45, two of the VR1055vapA-vaccinated foals had increased titers of VapA-specific IgGb, IgM and IgGa in the sera, and IgG in the BALF. The induction of the opsonizing isotypes IgGa and IgGb has been previously shown to be associated with protection against R. equi. No VapA-specific immune responses were detected in the control group. This study indicates that the DNA vaccine effectively stimulates anamnestic systemic and pulmonary immune responses in adult horses. The results in foals suggest that the DNA vaccine also primed a subset of immunized neonates. These data support further development and modification to produce a DNA vaccine to more effectively prime neonatal foals.

Genetic and antigenic characterization of Babesia bovis merozoite spherical body protein Bb-1 *

A Babesia bovis merozoite protein, Bb-1, was localized by immunoelectron microscopy to an apical organelle known as the spherical body. This unique structure appears to be analogous to dense granules of other apicomplexan protozoa. Similar to previously described dense granule proteins of Plasmodium spp., Bb-1 is secreted during or just after invasion of host erythrocytes and becomes associated with the cytoplasmic face of the infected cell. The amino terminal sequence of Bb-1 contains a predicted signal peptide and is similar to the amino terminus of another spherical body protein (BvVA1/225) which is also translocated to the erythrocyte membrane. Importantly, these two spherical body proteins are the major components of a protective fraction of B. bovis antigen. There is marked conservation of Bb-1 amino acid sequences and B-lymphocyte epitopes among geographic strains. However, a divergent Bb-1 allele (Bv80) in Australia strains encodes six regions of amino acid polymorphism, including a region of tetrapeptide repeats in the C-terminal half of the polypeptide. Two of the polymorphic regions map to previously defined Th1 epitopes on Bb-1.

Isolate-specific parasite antigens of the Babesia bovis-infected erythrocyte surface.

Bovine erythrocytes taken from in vitro cultures of Babesia bovis parasites from Mexico and the United States were assayed for the presence of new epitopes on the erythrocyte surface. New surface-exposed epitopes were detected by means of a whole-cell antigen capture assay. These epitopes were subsequently demonstrated only on infected erythrocytes by immunofluorescence staining of intact, living cells. Parasite-synthesized antigens were identified on each isolate using a surface-specific immunoprecipitation technique to analyze metabolically-labeled infected erythrocytes. In the Mexico isolate these antigens were 120 kDa and 107 kDa, whereas in the United States isolate polypeptides of 135, 120 and 107 kDa were detected. In each of these assays, reaction of immune sera with the infected erythrocyte surface was found to be isolate-specific.