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Trypanosoma vivaxis a unicellular hemoparasite, and a principal cause of animal African trypanosomiasis (AAT), a vector-borne and potentially fatal disease of livestock across sub-Saharan Africa. Previously, we identified diverseT. vivax-specific genes that were predicted to encode cell surface proteins. Here, we examine the immune responses of naturally and experimentally infected hosts to many of these unique parasite antigens, to identify immunogens that could become vaccine candidates. Immunoprofiling of host serum showed that one particular family (Fam34) elicits a consistent IgG antibody response. This gene family, which we now callVivaxin, encodes at least 124 transmembrane glycoproteins that display quite distinct expression profiles and patterns of genetic variation. We focused on one gene (viv-β8) that is among the most immunogenic and highly expressed but displays minimal polymorphism. VIVβ8 was localized across the cell body and flagellar membrane, suggesting that vivaxin is substantial family of novel surface proteins. Although vaccination of mice with VIVβ8 adjuvanted with Quil-A elicits a strong, balanced immune response and delays parasite proliferation in some animals, ultimately, it does not prevent disease. However, our phylogenetic analysis shows vivaxin includes other antigens shown to induce immunity againstT. vivax. Thus, the introduction of vivaxin represents an important advance in our understanding of theT. vivaxcell surface. Besides being a source of proven and promising vaccine antigens, the gene family is clearly an important component of the parasite glycocalyx, with potential to influence the host-parasite interaction.Author summaryAnimal African trypanosomiasis (AAT) is an important livestock disease throughout sub-Saharan Africa and beyond. AAT is caused by Trypanosoma vivax, among other species, a unicellular parasite that is spread by biting tsetse flies and multiplies in the bloodstream and other tissues, leading to often fatal neurological conditions if untreated. Although concerted drug treatment and vector eradication programmes have succeeded in controlling Human African trypanosomiasis, AAT continues to adversely affect animal health and impede efficient food production and economic development in many less-developed countries. In this study, we attempted to identify parasite surface proteins that stimulated the strongest immune responses in naturally infected animals, as the basis for a vaccine. We describe the discovery of a new, species-specific protein family in T. vivax, which we call vivaxin. We show that one vivaxin protein (VIVβ8) is surface expressed and retards parasite proliferation when used to immunize mice, but does not prevent infection. However, we also reveal that vivaxin includes another protein previously shown to induce protective immunity (IFX/VIVβ1). Besides its great potential for novel approaches to AAT control, vivaxin is revealed as a significant component of the T. vivax cell surface and may have important, species-specific roles in host interactions.

Trypanosomes are protozoan parasites that cause infectious diseases including human African trypanosomiasis (sleeping sickness), and nagana in economically-important livestock animals1,2. An effective vaccine against trypanosomes would be an important control tool, but the parasite has evolved sophisticated immunoprotective mechanisms including antigenic variation3 that present an apparently insurmountable barrier to vaccination. Here we show using a systematic genome-led vaccinology approach4 and a murine model of Trypanosoma vivax infection5 that protective invariant subunit vaccine antigens can be identified. Vaccination with a single recombinant protein comprising the extracellular region of a conserved cell surface protein localised to the flagellum membrane termed “invariant flagellum antigen from T. vivax” (IFX) induced long-lasting protection. Immunity was passively transferred with immune serum, and recombinant monoclonal antibodies to IFX could induce sterile protection and revealed multiple mechanisms of antibody-mediated immunity, including a major role for complement. Our discovery identifies a vaccine candidate for an important parasitic disease that has constrained the socioeconomic development of sub-Saharan African countries6 and challenges long-held views that vaccinating against trypanosome infections cannot be achieved.

Trypanosomes are protozoan parasites that cause infectious diseases, including African trypanosomiasis (sleeping sickness) in humans and nagana in economically important livestock1,2. An effective vaccine against trypanosomes would be an important control tool, but the parasite has evolved sophisticated immunoprotective mechanisms—including antigenic variation3—that present an apparently insurmountable barrier to vaccination. Here we show, using a systematic genome-led vaccinology approach and a mouse model of Trypanosoma vivax infection4, that protective invariant subunit vaccine antigens can be identified. Vaccination with a single recombinant protein comprising the extracellular region of a conserved cell-surface protein that is localized to the flagellum membrane (which we term ‘invariant flagellum antigen from T. vivax’) induced long-lasting protection. Immunity was passively transferred with immune serum, and recombinant monoclonal antibodies to this protein could induce sterile protection and revealed several mechanisms of antibody-mediated immunity, including a major role for complement. Our discovery identifies a vaccine candidate for an important parasitic disease that has constrained socioeconomic development in countries in sub-Saharan Africa5, and provides evidence that highly protective vaccines against trypanosome infections can be achieved. Systemic genome-led vaccinology and a mouse model of Trypanosoma vivax infection identify protective invariant subunit vaccine antigens, and demonstrate the possibility of generating effective vaccines that induce long-lasting protection against trypanosome infections.

The advances in microscopy combined to the invaluable progress carried by the utilization of molecular, immunological or immunochemical markers and the implementation of more powerful imaging technologies have yielded great improvements to the knowledge of the interaction between microorganisms and their hosts, notably a better understanding of the establishment of infectious processes. Still today, the intricacies of the dialog between parasites, cells and tissues remain limited. Some improvements have been attained with the stable integration and expression of the green fluorescence protein or firefly luciferase and other reporter genes, which have allowed to better approach the monitoring of gene expression and protein localization in vivo, in situ and in real time. Aiming at better exploring the well-established models of murine infections with the characterized strains of Trypanosoma cruzi and Trypanosoma vivax, we revisited in the present report the state of the art about the tools for the imaging of Trypanosomatids in vitro and in vivo and show the latest transgenic parasites that we have engineered in our laboratory using conventional transfection methods. The targeting of trypanosomes presented in this study is a promising tool for approaching the biology of parasite interactions with host cells, the progression of the diseases they trigger and the screening of new drugs in vivo or in vitro.

Perinatal asphyxia and other birth complications can lead to hypoxic–ischemic (HI) injury to the neonatal brain. Preconditioning is a stimulus that triggers endogenous neuroprotective mechanisms in the brain and thereby provides protection from subsequent injury (1). In neonatal mice, a brief episode of systemic hypoxia is an effective stimulus for triggering neuroprotective mechanisms that lead to a reduction in HI brain injury (2). Studying the mechanisms involved in hypoxic preconditioning (HPC) provides a unique opportunity to understand endogenous neuroprotective processes that could also be important for the injured brain (1). We have previously shown that HI in neonatal mice is associated with a marked decrease in glutathione peroxidase (GPx) activity 24 h after HI, while catalase activity remains unchanged (3). This led us to investigate whether overexpression of human GPx 1 (GPx1) protects the neonatal brain from HI injury (4). Similar to adult animals after focal ischemia (5), GPx1-overexpressing mice have reduced histological brain injury after HI, indicating that oxidative stress is a critical mediator of the injury response after neonatal HI. The protection afforded by GPx1 overexpression is associated with increased GPx activity and reduced hydrogen peroxide accumulation after HI (2). GPx1 overexpression also reduces HI injury in neonatal mice overexpressing human superoxide dismutase 1 (6), which have been shown to accumulate higher levels of hydrogen peroxide after HI (3) and present with greater damage as compared with nontransgenic wild-type (WT) littermates (7). These results clearly show that hydrogen peroxide plays an important role in neonatal HI injury and highlight the importance of GPx activity in removing hydrogen peroxide during HI. Paradoxically, however, we recently found that GPx1 overexpression reverses the neuroprotection afforded by HPC, which by itself does not affect GPx activity (2). This finding suggests that a certain threshold of hydrogen peroxide may be required during the preconditioning phase for the development of tolerance toward subsequent injury. Development of tolerance by HPC is a time-dependent process that requires activation of transcriptional events to achieve maximum protection. Although much emphasis has been given to hypoxia-inducible factor 1α (HIF-1α) as a mediator of this response, there is evidence that activation of extracellular signal-regulated kinase 1/2 (ERK1/2) or Akt may also be important in this process (8,9). Thus, using oxygen/glucose deprivation to mimic ischemia in vivo, it was shown that inhibition of the ERK 1/2 pathway abolished the protective effect of an ischemic preconditioning stimulus on the survival of isolated cortical rat neurons (10). More important, a study in neonatal rats showed that cortical ERK1/2 was transiently activated during the HPC phase, and inhibition of the ERK pathway with the MEK1/2 inhibitor U0126 reversed HPC protection (11). The role of Akt activation in HPC protection in vivo is more controversial, as preconditioning is often not accompanied by activation of Akt (11,12). Here, we assessed the temporal changes in the activation (phosphorylation) of ERK1/2 and Akt and the induction of HIF-1α in the cortex of mice subjected to HPC, and determined how these pathways are affected by GPx1 overexpression, as these pathways have been shown to be subject to redox control (13–15). We focused on the cortex because it is one of the main structures affected by HI in neonatal mice (16); it is protected by HPC (2) and a comparison with previously published data is also drawn (11). We show that HPC is associated with transient activation of ERK1/2 but not Akt in the cortex of WT animals, and that this activation occurs in the neurons, endothelial cells, and astrocytes in vulnerable regions and is associated with nuclear translocation. Finally, GPx1 overexpression blocked this activation and nuclear translocation of ERK, whereas it had no effect on the induction of HIF-1α. These results show that ERK activation in the murine brain by HPC is redox dependent and provides evidence that aberrant ERK activation is responsible for the paradoxical reversal of HPC protection in GPx1-overexpressing mice.

Animal African trypanosomiasis (AAT) is a severe, wasting disease of domestic livestock and diverse wildlife species. The disease in cattle kills millions of animals each year and inflicts a major economic cost on agriculture in sub-Saharan Africa. Cattle AAT is caused predominantly by the protozoan parasites Trypanosoma congolense and T. vivax, but laboratory research on the pathogenic stages of these organisms is severely inhibited by difficulties in making even minor genetic modifications. As a result, many of the important basic questions about the biology of these parasites cannot be addressed. Here we demonstrate that an in vitro culture of the T. congolense genomic reference strain can be modified directly in the bloodstream form reliably and at high efficiency. We describe a parental single marker line that expresses T. congolense-optimized T7 RNA polymerase and Tet repressor and show that minichromosome loci can be used as sites for stable, regulatable transgene expression with low background in non-induced cells. Using these tools, we describe organism-specific constructs for inducible RNA-interference (RNAi) and demonstrate knockdown of multiple essential and non-essential genes. We also show that a minichromosomal site can be exploited to create a stable bloodstream-form line that robustly provides >40,000 independent stable clones per transfection–enabling the production of high-complexity libraries of genome-scale. Finally, we show that modified forms of T. congolense are still infectious, create stable high-bioluminescence lines that can be used in models of AAT, and follow the course of infections in mice by in vivo imaging. These experiments establish a base set of tools to change T. congolense from a technically challenging organism to a routine model for functional genetics and allow us to begin to address some of the fundamental questions about the biology of this important parasite., Author summary The parasites Trypanosoma congolense and T. vivax are the most significant causative agents of Animal African trypanosomiasis (AAT). AAT kills an estimated 3 million cattle each year and represents a huge financial burden on food production in sub-Saharan Africa. A critical tool for understanding pathogen biology is the ability to make genetic modifications, especially creating specific mutants of target genes that can be used to investigate the locations of gene products, the effects of changes in expression, or consequence of complete gene removal. However, work on AAT is severely limited by difficulties in making even small genetic modifications and lack of tools for many functional genetics applications. Here, we design, test and validate a set of tools for T. congolense that brings for the first time: routine high-efficiency gene tagging and knockout, regulatable transgene expression from silent loci, a species-specific system for inducible gene knockdown, bioluminescent lines for in vivo disease models, and a means to generate highly complex libraries of mutants that will enable genome-scale work. These data and the tools around them will greatly aid research into AAT and T. congolense biology.