Role of Brucella Toll/Interleukin-1 Receptor (TIR) Domain Containing Protein (∆TcpB) Deletion Mutant in Protective Immunity against Brucellosis

Brucellosis is an important zoonotic disease affecting about 500,000 people annually. The development of safer and more efficacious Brucella Live attenuated vaccines addresses safety concerns that include the identification of reproducible and reliable surrogates of protection and mechanisms to bolster longevity. Brucella encodes a toll interleukin receptor domain containing protein (TcpB/Btp-1). These proteins subvert host innate immunity by abrogating NF-κB mediated cytokine production, by binding to and/or causing the degradation of signaling molecules TIRAP (MAL) and MyD88. TcpB has also been shown to directly reduce the CTL killing activity of infected host cells. In the current study, we investigated the effect of deleting tcpB from Brucella on invitro and invivo immune responses. We also evaluated an in vitro murine Brucella growth inhibition co-culture assay to determine the capacity of immune splenocytes from mice exposed to the tcpB mutant or wild type to control the growth of Brucella melitensis in murine bone marrow derived macrophages. A tcpB knockout constructed by gene replacement in the Brucella abortus S19 genetic background was used to vaccinate C57BL/6 mice assessed for development of CD4+ memory T cells. Mice vaccinated with the mutant displayed an elevated Th1 response, compared to the parental S19 and non- vaccinated controls, as manifested by multiple factors. These include; elevated IFN-γ early post vaccination, and a significant elevation of memory CD4+CD44+CD62L+ within CD4+T cell population in splenocytes derived from mice vaccinated with the mutant. S19 and S19ΔtcpB strains induced a significant increase in the IgG2a levels post vaccination. Consistent with a shift to a Th1 response, S19ΔtcpB induced a higher response later in vaccination. Splenocytes obtained from mice vaccinated with the S19ΔtcpB mutant exhibited significantly higher levels of killing activity compared to cells derived from S19 vaccinated mice and PBS controls. Consistent with enhanced immune protection, fewer bacteria were recovered from the spleens of mice vaccinated with the S19ΔtcpB mutant, which had reduced inflammatory lesions consistent with reduced bacterial burden. These results provide strong evidence that tcpB deletion improves immunogenicity, longevity and protective efficacy of S19 and that ex vivo co- cultivation may be employed to predict potential vaccine efficacy

Age-Related Differential Stimulation of Immune Response by Babesia microti and Borrelia burgdorferi During Acute Phase of Infection Affects Disease Severity

Lyme disease is the most prominent tick-borne disease with 300,000 cases estimated by CDC every year while ~2,000 cases of babesiosis occur per year in the United States. Simultaneous infection with Babesia microti and Borrelia burgdorferi are now the most common tick-transmitted coinfections in the U.S.A., and they are a serious health problem because coinfected patients show more intense and persisting disease symptoms. B. burgdorferi is an extracellular spirochete responsible for systemic Lyme disease while B. microti is a protozoan that infects erythrocytes and causes babesiosis. Immune status and spleen health are important for resolution of babesiosis, which is more severe and even fatal in the elderly and splenectomized patients. Therefore, we investigated the effect of each pathogen on host immune response and consequently on severity of disease manifestations in both young, and 30 weeks old C3H mice. At the acute stage of infection, Th1 polarization in young mice spleen was associated with increased IFN-γ and TNF-α producing T cells and a high Tregs/Th17 ratio. Together, these changes could help in the resolution of both infections in young mice and also prevent fatality by B. microti infection as observed with WA-1 strain of Babesia. In older mature mice, Th2 polarization at acute phase of B. burgdorferi infection could play a more effective role in preventing Lyme disease symptoms. As a result, enhanced B. burgdorferi survival and increased tissue colonization results in severe Lyme arthritis only in young coinfected mice. At 3 weeks post-infection, diminished pathogen-specific antibody production in coinfected young, but not older mice, as compared to mice infected with each pathogen individually may also contribute to increased inflammation observed due to B. burgdorferi infection, thus causing persistent Lyme disease observed in coinfected mice and reported in patients. Thus, higher combined proinflammatory response to B. burgdorferi due to Th1 and Th17 cells likely reduced B. microti parasitemia significantly only in young mice later in infection, while the presence of B. microti reduced humoral immunity later in infection and enhanced tissue colonization by Lyme spirochetes in these mice even at the acute stage, thereby increasing inflammatory arthritis

Protozoan parasite babesia microti subverts adaptive immunity and enhances lyme disease severity

Lyme disease is the most prominent tick-borne disease in the United States. Co-infections with the tick-transmitted pathogens Babesia microti and Borrelia burgdorferi sensu stricto are becoming a serious health problem. B. burgdorferi is an extracellular spirochete that causes Lyme disease while B. microti is a protozoan that infects erythrocytes and causes babesiosis. Testing of donated blood for Babesia species is not currently mandatory due to unavailability of an FDA approved test. Transmission of this protozoan by blood transfusion often results in high morbidity and mortality in recipients. Infection of C3H/HeJ mice with B. burgdorferi and B. microti individually results in inflammatory Lyme disease and display of human babesiosis-like symptoms, respectively. Here we use this mouse model to provide a detailed investigation of the reciprocal influence of the two pathogens on each other during coinfection. We show that B. burgdorferi infection attenuates parasitemia in mice while B. microti subverts the splenic immune response, such that a marked decrease in splenic B and T cells, reduction in antibody levels and diminished functional humoral immunity, as determined by spirochete opsonophagocytosis, are observed in co-infected mice compared to only B. burgdorferi infected mice. Furthermore, immunosuppression by B. microti in coinfected mice showed an association with enhanced Lyme disease manifestations. This study demonstrates the effect of only simultaneous infection by B. burgdorferi and B. microti on each pathogen, immune response and on disease manifestations with respect to infection by the spirochete and the parasite. In our future studies, we will examine the overall effects of sequential infection by these pathogens on host immune responses and disease outcomes. Copyright © 2019 Djokic, Akoolo, Primus, Schlachter, Kelly, Bhanot and Parveen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms

The Case for Live Attenuated Vaccines against the Neglected Zoonotic Diseases Brucellosis and Bovine Tuberculosis

Vaccination of humans and animals with live attenuated organisms has proven to be an effective means of combatting some important infectious diseases. In fact, the 20th century witnessed tremendous improvements in human and animal health worldwide as a consequence of large-scale vaccination programs with live attenuated vaccines (LAVs). Here, we use the neglected zoonotic diseases brucellosis and bovine tuberculosis (BTb) caused by Brucella spp. and Mycobacterium bovis (M. bovis), respectively, as comparative models to outline the merits of LAV platforms with emphasis on molecular strategies that have been pursued to generate LAVs with enhanced vaccine safety and efficacy profiles. Finally, we discuss the prospects of LAV platforms in the fight against brucellosis and BTb and outline new avenues for future research towards developing effective vaccines using LAV platforms

Babesia microti infection changes host spleen architecture and is cleared by a Th1 immune response

Babesia microti is a malaria-like parasite, which infects ~2000 people annually, such that babesiosis is now a notifiable disease in the United States. Immunocompetent individuals often remain asymptomatic and are tested only after they feel ill. Susceptible C3H/HeJ mice show several human-like disease manifestations and are ideal to study pathogenesis of Babesia species. In this study, we examined parasitemia of B. microti at different time points and assessed its impact on hemoglobin levels in blood, on spleen pathology and overall immune response in C3H/HeJ mice. Peak parasitemia of 42.5% was immediately followed by diminished hemoglobin level. Parasitemia at 21 days of infection was barely detectable by microscopy presented 5.7 × 108 to 5.9 × 109 B. microti DNA copies confirming the sensitivity of our qPCR. We hypothesize that qPCR detects DNA released from recently lysed parasites or from extracellular B. microti in blood, which are not easily detected in blood smears and might result in under-diagnosis of babesiosis in patients. Splenectomized patients have been reported to show increased babesiosis severity and result in high morbidity and mortality. These results emphasize the importance of splenic immunity in resolution of B. microti infection. Splenomegaly in infected mice associated with destruction of marginal zone with lysed erythrocytes and released B. microti life forms in our experiments support this premise. At conclusion of the experiment at 21 days post-infection, significant splenic B and T cells depletion and increase in macrophages levels were observed in B. microti infected mice suggesting a role of macrophage in disease resolution. Infected mice also showed significantly higher plasmatic concentration of CD4 Th1 cells secreted cytokines such as IL-2 and IFN-γ while cytokines such as IL-4, IL-5, and IL-13 secreted by Th2 cells increase was not always significant. Thus, Th1 cells-mediated immunity appears to be important in clearance of this intracellular pathogen. Significant increase in IL-6 that promotes differentiation of Th17 cells was observed but it resulted in only moderate change in IL-17A, IL-17F, IL-21, and IL-22, all secreted by Th17 cells. A similar immune response to Trypanosoma infection has been reported to influence the clearance of this protozoan, and co-infecting pathogen(s)

Two Theileria parva CD8 T Cell Antigen Genes Are More Variable in Buffalo than Cattle Parasites, but Differ in Pattern of Sequence Diversity

<p><b>Background:</b> Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8(+) T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8(+) T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8(+) T-cell epitopes, and to analyse the sequences for evidence of selection.</p> <p><b>Methodology/Principal Findings:</b> Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (similar to 12%) in Tp1 and in 320 positions (similar to 61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle.</p> <p><b>Conclusions/Significance:</b> The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.</p&gt

Two Theileria parva CD8 T Cell Antigen Genes Are More Variable in Buffalo than Cattle Parasites, but Differ in Pattern of Sequence Diversity

<p><b>Background:</b> Theileria parva causes an acute fatal disease in cattle, but infections are asymptomatic in the African buffalo (Syncerus caffer). Cattle can be immunized against the parasite by infection and treatment, but immunity is partially strain specific. Available data indicate that CD8(+) T lymphocyte responses mediate protection and, recently, several parasite antigens recognised by CD8(+) T cells have been identified. This study set out to determine the nature and extent of polymorphism in two of these antigens, Tp1 and Tp2, which contain defined CD8(+) T-cell epitopes, and to analyse the sequences for evidence of selection.</p> <p><b>Methodology/Principal Findings:</b> Partial sequencing of the Tp1 gene and the full-length Tp2 gene from 82 T. parva isolates revealed extensive polymorphism in both antigens, including the epitope-containing regions. Single nucleotide polymorphisms were detected at 51 positions (similar to 12%) in Tp1 and in 320 positions (similar to 61%) in Tp2. Together with two short indels in Tp1, these resulted in 30 and 42 protein variants of Tp1 and Tp2, respectively. Although evidence of positive selection was found for multiple amino acid residues, there was no preferential involvement of T cell epitope residues. Overall, the extent of diversity was much greater in T. parva isolates originating from buffalo than in isolates known to be transmissible among cattle.</p> <p><b>Conclusions/Significance:</b> The results indicate that T. parva parasites maintained in cattle represent a subset of the overall T. parva population, which has become adapted for tick transmission between cattle. The absence of obvious enrichment for positively selected amino acid residues within defined epitopes indicates either that diversity is not predominantly driven by selection exerted by host T cells, or that such selection is not detectable by the methods employed due to unidentified epitopes elsewhere in the antigens. Further functional studies are required to address this latter point.</p&gt

Efficient detection of symptomatic and asymptomatic patient samples for Babesia microti and Borrelia burgdorferi infection by multiplex qPCR.

BACKGROUND:Tick-borne infections have been increasing steadily over the years, with co-infections with Borrelia burgdorferi and Babesia microti/divergens emerging as a serious health problem. B. burgdorferi is a spirochetal bacterium that causes Lyme disease while protozoan pathogens belonging to Babesia species are responsible for babesiosis. Currently used serological tests do not always detect acute Lyme disease or babesiosis, and fail to differentiate cured patients from those who get re-infected. This is a major problem for proper diagnosis particularly in regions endemic for tick-borne diseases. Microscopy based evaluation of babesiosis is confirmatory but is labor intensive and insensitive such that many asymptomatic patients remain undetected and donate blood resulting in transfusion transmitted babesiosis. RESULTS:We conducted multiplex qPCR for simultaneous diagnosis of active Lyme disease and babesiosis in 192 blood samples collected from a region endemic for both diseases. We document qPCR results obtained from testing of each sample three times to detect infection with each pathogen separately or together. Results for Lyme disease by qPCR were also compared with serological tests currently used for Lyme disease when available. Considering at least two out of three test results for consistency, 18.2% of patients tested positive for Lyme disease, 18.7% for co-infection with B. burgdorferi and B. microti and 6.3% showed only babesiosis. CONCLUSIONS:With an 80% sensitivity for detection of Lyme disease, and ability to detect co-infection with B. microti, multiplex qPCR can be employed for diagnosis of these diseases to start appropriate treatment in a timely manner

Evaluation of the recognition of Theileria parva vaccine candidate antigens by cytotoxic T lymphocytes from Zebu cattle.

East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated

Evaluation of the recognition of Theileria parva vaccine candidate antigens by cytotoxic T lymphocytes from Zebu cattle.

East Coast fever (ECF) is a highly fatal lymphoproliferative disease of cattle caused by Theileria parva, a tick-borne intracellular apicomplexan parasite. Parasite antigens that are targets of protective cytotoxic T lymphocyte (CTL) responses are required to formulate a sub-unit vaccine against ECF. A number of CTL target antigens have recently been identified and initial evaluation has shown their vaccine potential. This study aimed to evaluate whether these antigens were recognised by CTL obtained from six genetically diverse Zebu cattle immunized with a cocktail of T. parva stocks. T. parva Muguga specific polyclonal CD8(+) CTL lines were generated and confirmed to specifically lyse autologous infected cells. CTL recognition of autologous skin fibroblasts (iSF) transduced with recombinant modified vaccinia virus Ankara strain (MVA) expressing previously identified T. parva Muguga vaccine candidate antigens was evaluated using an IFN-gamma ELISpot assay. CTL lines from one of the four calves, BY120, responded specifically to cells infected with MVA expressing the antigen Tp2 and synthetic peptides were employed to map a new CTL epitope on this antigen. Immunoscreening of the T. parva genome with these CTL lines should identify novel antigens that will constitute valuable additions to the vaccine candidates currently being evaluated