Neutralizing antibody-independent immunity to SARS-CoV-2 in hamsters and hACE-2 transgenic mice immunized with a RBD/Nucleocapsid fusion protein [preprint]

The nucleocapsid (N) and the receptor binding domain (RBD) of the Spike (S) proteins elicit robust antibody and T cell responses either in vaccinated or COVID-19 convalescent individuals. We generated a chimeric protein that comprises the sequences of RBD from S and N antigens (SpiN). SpiN was highly immunogenic and elicited a strong IFNγ response from T cells and high levels of antibodies to the inactivated virus, but no neutralizing antibodies. Importantly, hamsters and the human Angiotensin Convertase Enzyme-2-transgenic mice immunized with SpiN were highly resistant to challenge with the wild type SARS-CoV-2, as indicated by viral load, clinical outcome, lung inflammation and lethality. Thus, the N protein should be considered to induce T-cell-based immunity to improve SARS-CoV-2 vaccines, and eventually to circumvent the immune scape by variants

Trypanosoma cruzi and its components as exogenous mediators of inflammation recognized through Toll-like receptors.

Trypanosoma cruzi is the etiologic agent of Chagas' disease, a parasitic disease of enormous importance in Latin America. Herein we review the studies that revealed the receptors from innate immunity that are involved in the recognition of this protozoan parasite. We showed that the recognition of T. cruzi and its components occurs through Toll-like receptors (TLR) 2/CD14. Further, we showed in vivo the importance of the myeloid differentiation factor (MyD88), an adapter protein essential for the function of TLRs, in determining the parasitemia and mortality rate of mice infected with T. cruzi. We also discuss the implications of these findings in the pathophysiology of Chagas' disease

Protective immunization against murine cytomegalovirus infection using adenoviruses and poxviruses expressing hepatitis B virus chimeras

Recombinant adenoviruses, poxviruses, and plasmid DNA vaccines encoding different hepatitis B virus (HBV)/murine cytomegalovirus (MCMV) protein chimeras were used to immunize mice. Processing of the chimeras resulted in presentation of a protective Ld/CD8+ T-cell epitope of the immediate early 1 protein pp89 (IE1 pp89) of MCMV to the immune system. Different levels of immunogenicity were observed depending on: (i) the type of viral vector used, (ii) whether the antigens were included in the cellular secretion pathway, and (iii) the location of the protective epitope within the chimeric protein. An adenovirus expressing a secretory HBV core protein with the MCMV epitope in its C-terminus induced the highest immune response. When the most immunogenic adenovirus and vaccinia virus were used in a heterologous prime-boost immunization protocol, even higher levels of epitope-specific T cells were obtained. Furthermore, responses were protective against a challenge with MCMV, inducing up to a 96% reduction of viral load in immunized animals, as determined by a sensitive real-time PCR assay. Together, these results confirmed previous observations of the efficient use of adenoviral and poxviral vectors in prime-boost protocols for immunization against diseases whose resolution depends on cellular immunity, as well as the aptness of correctly designed chimeric carrier proteins to facilitate this goal. [Int Microbiol 2007; 10(4):261-269

Leishmania amazonensis Engages CD36 to Drive Parasitophorous Vacuole Maturation

Leishmania amastigotes manipulate the activity of macrophages to favor their own success. However, very little is known about the role of innate recognition and signaling triggered by amastigotes in this host-parasite interaction. In this work we developed a new infection model in adult Drosophila to take advantage of its superior genetic resources to identify novel host factors limiting Leishmania amazonensis infection. The model is based on the capacity of macrophage-like cells, plasmatocytes, to phagocytose and control the proliferation of parasites injected into adult flies. Using this model, we screened a collection of RNAi-expressing flies for anti-Leishmania defense factors. Notably, we found three CD36-like scavenger receptors that were important for defending against Leishmania infection. Mechanistic studies in mouse macrophages showed that CD36 accumulates specifically at sites where the parasite contacts the parasitophorous vacuole membrane. Furthermore, CD36-deficient macrophages were defective in the formation of the large parasitophorous vacuole typical of L. amazonensis infection, a phenotype caused by inefficient fusion with late endosomes and/or lysosomes. These data identify an unprecedented role for CD36 in the biogenesis of the parasitophorous vacuole and further highlight the utility of Drosophila as a model system for dissecting innate immune responses to infection

Use of whole plant Artemisia annua L. as an antimalarial therapy

Anti-malarial drugs are primary weapons for reducing Plasmodium transmission in human populations. Successful drugs have been highly efficacious and inexpensive to synthetically manufacture. Emergence of resistant parasites reduces the lifespan of each drug that is developed and deployed. Currently, the most effective anti-malarial is artemisinin (AN), which is extracted from the leaves of Artemisia annua. Because of its poor pharmacokinetic properties and prudent efforts to curtail emergence of resistance, AN is prescribed only in combination with other anti-malarials composing an Artemisinin Combination Therapy (ACT). Low yield in the plant and the added cost of secondary anti-malarials in the ACT, make AN in the developing world a costly treatment. Here we show that dried leaves of A. annua administered orally are more effective at killing malaria parasites than a comparable dose of purified drug in a rodent malaria model (P. chabaudi). A single dose of whole plant (WP) A. annua containing 24 mg/kg AN clears 99% of parasites, where a comparable dose of pure drug has half that effect. This is consistent with findings that blood levels of AN are 40 times greater in mice receiving WP versus those given pure drug. We hypothesize that in addition to increasing bioavailability of AN, administration of WP alone may constitute a combination therapy because it contains other anti-malarial compounds that have been shown to synergize with AN. Inexpensive, efficacious, and resilient treatment for malaria based upon WP A. annua that can be grown and processed locally would be an effective addition to the global effort to reduce malaria morbidity and mortality

Increased survival in B-cell-deficient mice during experimental cerebral malaria suggests a role for circulating immune complexes

The pathogenesis of malaria, an insect-borne disease that takes millions of lives every year, is still not fully understood. Complement receptor 1 (CR1) has been described as a receptor for Plasmodium falciparum, which causes cerebral malaria in humans. We investigated the role of CR1 in an experimental model of cerebral malaria. Transgenic mice expressing human CR1 (hCR1(+)) on erythrocytes were infected with Plasmodium berghei ANKA and developed cerebral malaria. No difference in survival was observed in hCR1(+) mice compared to wild-type mice following infection with P. berghei ANKA; however, hCR1 detection was significantly diminished on erythrocytes between days 7 and 10 postinfection. hCR1 levels returned to baseline by day 17 postinfection in surviving animals. Immunoblot assays revealed that total erythrocyte hCR1 levels were diminished, confirming that immune complexes in association with erythrocyte hCR1 were likely removed from erythrocytes in vivo by clearance following immune adherence. Decreases in hCR1 were completely dependent on C3 expression, as mice treated with cobra venom factor (which consumes and depletes C3) retained hCR1 on erythrocytes during C3 depletion through day 7; erythrocyte hCR1 decreases were observed only when C3 levels recovered on day 9. B-cell-deficient mice exhibit a marked increase in survival following infection with P. berghei ANKA, which suggests that immune complexes play a central role in the pathogenesis of experimental cerebral malaria. Together, our findings highlight the importance of complement and immune complexes in experimental cerebral malaria. IMPORTANCE Cerebral malaria is a deadly complication of infection with Plasmodium falciparum. Despite its high prevalence, relatively little is understood about its pathogenesis. We have determined that immune complexes are generated and deposited on erythrocytes specifically expressing human complement receptor 1 in a mouse model of cerebral malaria. We also provide evidence demonstrating the importance of immunoglobulins in the pathogenesis of cerebral malaria in mice. These findings may have important implications in human cerebral malaria

Killer lymphocytes use granulysin, perforin and granzymes to kill intracellular parasites

Protozoan infections are a serious global health problem1, 2. Natural killer (NK) cells and cytolytic T lymphocytes (CTLs) eliminate pathogen-infected cells by releasing cytolytic granule contents—granzyme (Gzm) proteases and the pore-forming perforin (PFN)—into the infected cell3. However, these cytotoxic molecules do not kill intracellular parasites. CD8+ CTLs protect against parasite infections in mice primarily by secreting interferon (IFN)-γ4, 5, 6, 7, 8, 9, 10. However, human, but not rodent, cytotoxic granules contain the antimicrobial peptide granulysin (GNLY), which selectively destroys cholesterol-poor microbial membranes11, 12, 13, 14, and GNLY, PFN and Gzms rapidly kill intracellular bacteria15. Here we show that GNLY delivers Gzms into three protozoan parasites (Trypanosoma cruzi, Toxoplasma gondii and Leishmania major), in which the Gzms generate superoxide and inactivate oxidative defense enzymes to kill the parasite. PFN delivers GNLY and Gzms into infected cells, and GNLY then delivers Gzms to the intracellular parasites. Killer cell–mediated parasite death, which we term 'microbe-programmed cell death' or 'microptosis', is caspase independent but resembles mammalian apoptosis, causing mitochondrial swelling, transmembrane potential dissipation, membrane blebbing, phosphatidylserine exposure, DNA damage and chromatin condensation. GNLY-transgenic mice are protected against infection by T. cruzi and T. gondii, and survive infections that are lethal to wild-type mice. Thus, GNLY-, PFN- and Gzm-mediated elimination of intracellular protozoan parasites is an unappreciated immune defense mechanism

Integrative analysis of microRNA and mRNA expression profiles of monocyte- derived dendritic cells differentiation during experimental cerebral malaria

Heterogeneity and high plasticity are common features of cells from the mononuclear phagocyte system: monocytes (MOs), macrophages, and dendritic cells (DCs). Upon activation by microbial agents, MO can differentiate into MO- derived DCs (MODCs). In previous work, we have shown that during acute infection with Plasmodium berghei ANKA (PbA), MODCs become, transiently, the main CD11b+ myeloid population in the spleen (SP) and once recruited to the brain play an important role in the development of experimental cerebral malaria (ECM). Here, we isolated 4 cell populations: bone marrow (BM) MOs (BM- MOs) and SP- MOs from uninfected mice; BM inflammatory MOs (BM- iMOs) and SP- MODCs from PbA- infected mice and used a system biology approach to a holistic transcriptomic comparison and provide an interactome analysis by integrating differentially expressed miRNAs (DEMs) and their differentially expressed gene targets (DEGs) data. The Jaccard index (JI) was used for gauging the similarity and diversity among these cell populations. Whereas BM- MOs, BM- iMOs, and SP- MOs presented high similarity of DEGs, SP- MODCs distinguished by showing a greater number of DEGs. Moreover, functional analysis identified an enrichment in canonical pathways, such as DC maturation, neuroinflammation, and IFN signaling. Upstream regulator analysis identified IFNγ as the potential upstream molecule that can explain the observed DEMs- Target DEGs intersections in SP- MODCs. Finally, directed target analysis and in vivo/ex vivo assays indicate that SP- MODCs differentiate in the SP and IFNγ is a main driver of this process.Graphical AbstractInteractome analysis between miRNAs and their target genes in IFNγ- mediated differentiation of splenic MODCs during Plasmodium infection.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/162711/1/jlb10625-sup-0002-TableS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162711/6/jlb10625-sup-0001-FigureS1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162711/5/jlb10625.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162711/4/jlb10625_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162711/3/jlb10625-sup-0004-TableS3.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162711/2/jlb10625-sup-0003-TableS2.pd

Contributions of IFN-gamma and granulysin to the clearance of Plasmodium yoelii blood stage

P. vivax-infected Retics (iRetics) express human leukocyte antigen class I (HLA-I), are recognized by CD8+ T cells and killed by granulysin (GNLY) and granzymes. However, how Plasmodium infection induces MHC-I expression on Retics is unknown. In addition, whether GNLY helps control Plasmodium infection in vivo has not been studied. Here, we examine these questions using rodent infection with the P. yoelii 17XNL strain, which has tropism for Retics. Infection with P. yoelii caused extramedullary erythropoiesis, reticulocytosis and expansion of CD8+CD44+CD62L- IFN-gamma-producing T cells that form immune synapses with iRetics. We now provide evidence that MHC-I expression by iRetic is dependent on IFN-gamma-induced transcription of IRF-1, MHC-I and beta2-microglobulin (beta2-m) in erythroblasts. Consistently, CTLs from infected wild type (WT) mice formed immune synapses with iRetics in an IFN-gamma- and MHC-I-dependent manner. When challenged with P. yoelii 17XNL, WT mice cleared parasitemia and survived, while IFN-gamma KO mice remained parasitemic and all died. beta2-m KO mice that do not express MHC-I and have virtually no CD8+ T cells had prolonged parasitemia, and 80% survived. Because mice do not express GNLY, GNLY-transgenic mice can be used to assess the in vivo importance of GNLY. Parasite clearance was accelerated in GNLY-transgenic mice and depletion of CD8+ T cells ablated the GNLY-mediated resistance to P. yoelii. Altogether, our results indicate that in addition to previously described mechanisms, IFN-gamma promotes host resistance to the Retic-tropic P. yoelii 17XNL strain by promoting MHC-I expression on iRetics that become targets for CD8+ cytotoxic T lymphocytes and GNLY