Job E. Lopez

Identification of Novel Antigenic Proteins in a Complex Anaplasma marginale Outer Membrane Immunogen by Mass Spectrometry and Genomic Mapping

ABSTRACT Immunization with purified Anaplasma marginale outer membranes induces complete protection against infection that is associated with CD4+ T-lymphocyte-mediated gamma interferon secretion and immunoglobulin G2 (IgG2) antibody titers. However, knowledge of the composition of the outer membrane immunogen is limited. Recent sequencing and annotation of the A. marginale genome predicts at least 62 outer membrane proteins (OMP), enabling a proteomic and genomic approach for identification of novel OMP by use of IgG serum antibody from outer membrane vaccinates. Outer membrane proteins were separated by two-dimensional electrophoresis, and proteins recognized by total IgG and IgG2 in immune sera of outer membrane-vaccinated cattle were detected by immunoblotting. Immunoreactive protein spots were excised and subjected to liquid chromatography-tandem mass spectrometry. A database search of the A. marginale genome identified 24 antigenic proteins that were predicted to be outer membrane, inner membrane, or membrane-associated proteins. These included the previously characterized surface-exposed outer membrane proteins MSP2, operon associated gene 2 (OpAG2), MSP3, and MSP5 as well as recently identified appendage-associated proteins. Among the 21 newly described antigenic proteins, 14 are annotated in the A. marginale genome and include type IV secretion system proteins, elongation factor Tu, and members of the MSP2 superfamily. The identification of these novel antigenic proteins markedly expands current understanding of the composition of the protective immunogen and provides new candidates for vaccine development.

Immunogenicity of Anaplasma marginale Type IV Secretion System Proteins in a Protective Outer Membrane Vaccine

ABSTRACT Rickettsial pathogens in the genera Anaplasma and Ehrlichia cause acute infection in immunologically naive hosts and are major causes of tick-borne disease in animals and humans. Immunization with purified outer membranes induces protection against acute Anaplasma marginale infection and disease, and a proteomic and genomic approach recently identified 21 proteins within the outer membrane immunogen in addition to the well-characterized major surface proteins MSP1 to MSP5. Among the newly described proteins were the type IV secretion system (TFSS) proteins VirB9, VirB10, and conjugal transfer protein (CTP). In other gram-negative bacteria, TFSS proteins form channels, facilitate secretion of molecules, and are required for intracellular survival. However, TFSS proteins have not been explored as vaccine antigens. In this study we demonstrate that in Anaplasma marginale outer membrane-vaccinated cattle, VirB9, VirB10, and CTP are recognized by serum immunoglobulin G2 (IgG2) and stimulate memory T-lymphocyte proliferation and gamma interferon secretion. VirB9 induced the greatest proliferation in CD4+ T-cell lines, and VirB9-specific CD4+ T-cell clones responded to three A. marginale strains, confirming the VirB9-specific T-cell responses are directed against epitopes in the native protein. The three TFSS proteins are highly conserved with orthologous proteins in Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia canis. Recognition of TFSS antigens by CD4+ T cells and by IgG2 from cattle immunized with the protective outer membrane fraction provides a rationale for including these proteins in development of vaccines against A. marginale and related pathogens.

Detection of Lassa Virus, Mali

To determine whether Lassa virus was circulating in southern Mali, we tested samples from small mammals from 3 villages, including Soromba, where in 2009 a British citizen probably contracted a lethal Lassa virus infection. We report the isolation and genetic characterization of Lassa virus from an area previously unknown for Lassa fever.

Anaplasma marginale Type IV Secretion System Proteins VirB2, VirB7, VirB11, and VirD4 Are Immunogenic Components of a Protective Bacterial Membrane Vaccine

ABSTRACT Anaplasma and related Ehrlichia spp. are important tick-borne, Gram-negative bacterial pathogens of livestock and humans that cause acute infection and disease and can persist. Immunization of cattle with an Anaplasma marginale fraction enriched in outer membranes (OM) can provide complete protection against disease and persistent infection. Serological responses of OM vaccinees to the OM proteome previously identified over 20 antigenic proteins, including three type IV secretion system (T4SS) proteins, VirB9-1, VirB9-2, and VirB10. Subsequent studies showed that these three proteins also stimulated CD4+ T-cell responses in OM vaccinees. The T4SS, composed of a complex of proteins spanning the inner and outer membranes of certain bacteria, is an important virulence factor but is relatively unexplored as a vaccine target. The goal of this study was to determine if additional T4SS proteins are immunogenic for animals immunized with the protective OM fraction of A. marginale. T4SS proteins expressed by in vitro transcription and translation were screened for stimulating proliferation of T cells from OM vaccinees, and immunogenic proteins were expressed as recombinant proteins in Escherichia coli and their immunogenicity was verified. VirB2, a putative VirB7, VirB11, and VirD4 were immunogenic for OM vaccinees expressing several common major histocompatibility complex (MHC) class II haplotypes. VirB2 is encoded by multiple genes that share a conserved central region, and epitope mapping revealed T-cell epitopes in this region. The discovery of novel immunogenic T4SS proteins recognized by outbred individuals with common MHC haplotypes further justifies evaluating the T4SS as a potential vaccine candidate for pathogenic bacteria.

Geographic Distribution and Genetic Characterization of Lassa Virus in Sub-Saharan Mali

Background Lassa fever is an acute viral illness characterized by multi-organ failure and hemorrhagic manifestations. Lassa fever is most frequently diagnosed in Nigeria, Sierra Leone, Liberia, and Guinea, although sporadic cases have been recorded in other West African countries, including Mali. The etiological agent of Lassa fever is Lassa virus (LASV), an Arenavirus which is maintained in nature and frequently transmitted to humans by Mastomys natalensis. The purpose of this study was to better define the geographic distribution of LASV-infected rodents in sub-Saharan Mali. Methodologies/Principal Findings Small mammals were live-trapped at various locations across Mali for the purpose of identifying potential zoonotic pathogens. Serological and molecular assays were employed and determined LASV infected rodents were exclusively found in the southern Mali near the border of Côte dIvoire. Overall, 19.4% of Mastomys natalensis sampled in this region had evidence of LASV infection, with prevalence rates for individual villages ranging from 0 to 52%. Full-length genomic sequences were determined using high throughput sequencing methodologies for LASV isolates generated from tissue samples of rodents collected in four villages and confirmed the phylogenetic clustering of Malian LASV with strain AV. Conclusions/Significance The risk of human infections with LASV is greatest in villages in southern Mali. Lassa fever should be considered in the differential diagnosis for febrile individuals and appropriate diagnostic techniques need to be established to determine the incidence of infection and disease in these regions.

Bloodmeal Size and Spirochete Acquisition of Ornithodoros hermsi (Acari: Argasidae) During Feeding

ABSTRACT Ornithodoros hermsi Wheeler (Acari: Argasidae) is the vector of Borrelia hermsii, the primary cause of tick-borne relapsing fever in North America. This tick is one of the smallest Ornithodoros species involved with the biological transmission of spirochetes; yet, the amount of blood ingested while feeding is unknown. Therefore, we determined the amount of blood O. hermsi ingested during a bloodmeal to establish its potential for spirochete acquisition while feeding on an infected host. Ticks at different developmental stages were weighed before and after feeding and the volume of blood ingested was calculated. Females ingested the most blood, averaging ≈15 µl per meal, but late-stage nymphs took in the most blood in proportion to unfed body weight. A cohort of nymphs was weighed three more times during the 48 h after feeding, which demonstrated that O. hermsi may have excreted coxal fluid ranging from 24–36% of the bloodmeal weight. We also developed a quantitative polymerase chain reaction method to determine the number of spirochetes ingested and maintained within the ticks after feeding. The density of spirochetes in ticks having just engorged was slightly less than in the hosts blood. In the first 5 d after feeding, the number of spirochetes within the ticks declined from the number initially ingested but then remained constant through 15 d. These observations establish a basis for future studies to determine the minimum number of spirochetes required in the hosts blood to allow O. hermsi to become persistently infected and transmit during subsequent bloodmeals.

Relapsing Fever Spirochetes Retain Infectivity After Prolonged in vitro Cultivation

Borrelia hermsii and Borrelia burgdorferi, two closely related spirochetes, are the etiological agents of tick-borne relapsing fever and Lyme disease, respectively. Previous studies have shown the loss of infectivity of B. burgdorferi is associated with in vitro cultivation. This diminished infectivity of B. burgdorferi has occurred as early as three in vitro passages, and the loss of plasmids have been observed with these less virulent to noninfective cultures. The effects of long-term in vitro cultivation on B. hermsii have not been investigated. However, understanding the degree of genomic degradation during in vitro cultivation is important for investigating pathogenic mechanisms of spirochetes. In this study, we analyzed the effects of continuous in vitro cultivation on the genomic composition and infectivity of B. hermsii and B. turicatae.We report that all seven isolates of B. hermsii and the one isolate of B. turicatae examined retained infectivity in mice after 1 year of continuous in vitro cultivation. Furthermore, there were few apparent differences in the plasmid profiles after long-term cultivation. Two isolates of B. hermsii remained infective after high passage despite losing a portion of the 200-kb linear plasmid containing the fhbA gene encoding the factor H binding protein. Also, sequence analysis of multiple B. hermsii isolates demonstrated two types of fhbA with complete congruence with the two genomic groups of B. hermsii spirochetes. Therefore, these results suggest that relapsing fever spirochetes are genetically stable during in vitro cultivation, and the fhbA-containing segment of DNA that is lost during cultivation is not required for infection.

Identification of conserved antigens for early serodiagnosis of relapsing fever Borrelia

Borrelia hermsii is a blood-borne pathogen transmitted by the argasid tick Ornithodoros hermsi. Since spirochaete clearance in mice is associated with an IgM-mediated response, an immunoproteomic analysis was used to identify proteins reactive with IgM. We report that IgM from both mice and human patients infected with B. hermsii not only reacted with the previously identified variable membrane proteins but also identified candidate antigens including heat-shock proteins, an adhesin protein, ABC transporter proteins, flagellar proteins, housekeeping proteins, an immune evasion protein, and proteins with unknown function. Furthermore, IgM reactivity to recombinant glycerophosphodiester phosphodiesterase was detected during early spirochaete infection and prior to a detectable IgG response. Lastly, a conserved hypothetical protein was produced in Escherichia coli and tested with immune serum against B. hermsii and Borrelia recurrentis. These results identify a much larger set of immunoreactive proteins, and could help in the early serodiagnosis of this tick-borne infection.

A Novel Surface Antigen of Relapsing Fever Spirochetes Can Discriminate between Relapsing Fever and Lyme Borreliosis

ABSTRACT In a previous immunoproteome analysis of Borrelia hermsii, candidate antigens that bound IgM antibodies from mice and patients infected with relapsing fever spirochetes were identified. One candidate that was identified is a hypothetical protein with a molecular mass of 57 kDa that we have designated Borrelia immunogenic protein A (BipA). This protein was further investigated as a potential diagnostic antigen for B. hermsii given that it is absent from the Borrelia burgdorferi genome. The bipA locus was amplified and sequenced from 39 isolates of B. hermsii that had been acquired from western North America. bipA was also expressed as a recombinant fusion protein. Serum samples from mice and patients infected with B. hermsii or B. burgdorferi were used to confirm the immunogenicity of the recombinant protein in patients infected with relapsing fever spirochetes. Lastly, in silico and experimental analysis indicated that BipA is a surface-exposed lipoprotein in B. hermsii. These findings enhance the capabilities of diagnosing infection with relapsing fever spirochetes.

Mesoamerican nephropathy: a neglected tropical disease with an infectious etiology?

An outbreak of unexplained and severe kidney disease, Mesoamerican Nephropathy, in mostly young, male sugar cane workers emerged in Central America in the late 1990s. As a result, an estimated 20,000 individuals have died, to date. Unfortunately, and with great consequence to human life, the etiology of the outbreak has yet to be identified. The sugarcane fields in Chichigalpa, Chinandega, Nicaragua, have been involved in the outbreak, and during our initial investigation, we interviewed case patients who experienced fever, nausea and vomiting, arthralgia, myalgia, headache, neck and back pain, weakness, and paresthesia at the onset of acute kidney disease. We also observed a heavy infestation of rodents, particularly of Sigmodon species, in the sugarcane fields. We hypothesize that infectious pathogens are being shed through the urine and feces of these rodents, and workers are exposed to these pathogens during the process of cultivating and harvesting sugarcane. In this paper, we will discuss the epidemic in the Chichigalpa area, potential pathogens responsible for Mesoamerican Nephropathy, and steps needed in order to diagnose, treat, and prevent future cases from occurring.