CD47 is an antiphagocytic (“don’t eat me”) signal expressed on all cells that inhibits programmed cell removal of self, and loss of this molecule by aging erythrocytes is associated with increased susceptibility to clearance by macrophages. Previously, we have demonstrated that absence of CD47 confers resistance to infection with nonlethal murine malaria Plasmodium yoelii 17XNL. Furthermore, CD47 blockade with an anti-CD47 monoclonal antibody promotes survival and reduces pathologic features of experimental cerebral malaria (ECM) during infection with lethal murine malaria P. berghei ANKA (Pb-A). To further define the immunological basis of CD47 regulation of ECM, we compared Pb-A infection in wild-type (WT) versus CD47 genetic knockout (CD47−/−) mice on the C57BL/6 background. Absence of CD47 resulted in partial but significant (log-rank P < 0.001) resistance to ECM and was associated with diminished sequestration of natural killer (NK) cells, CD4+ T cells, and CD8+ T cells (immune cell subsets essential for ECM pathogenesis) within the brain and lower levels of circulating interleukin-10 and MIP1β cytokines. Most importantly, our data show a critical role for CD47 in the maturation, activation, and recruitment of brain-sequestered NK cells. In mice experiencing ECM, CD47 associates with the exclusive expression of Ly49h (an activation receptor that elicits IFN-γ and cytolytic activity upon stimulation) by a subset of brain-sequestered CD11b+CD49b+ NK cells. Our studies provide insights on a previously undefined role of CD47 in NK cell–mediated pathogenesis of ECM and an increased understanding of the role of CD47 in NK cell biology.

The immunology of human babesiosis is poorly investigated. We present a comprehensive investigation of a 75-year-old man with B-cell deficiency who experienced 3 episodes of babesiosis over a 6-year period. Slowly evolving clinical immunity was observed, as evidenced by milder clinical symptoms and lower peak parasite burden after each subsequent babesiosis episode. The patient exhibited several striking immunologic findings. First, the patient had exceptionally high Babesia microti-specific antibodies despite very few circulating B cells, which predominantly coexpressed CD27 (memory marker) and CD95 (death receptor). Second, we demonstrated the presence of long-lasting NK cells and expansion of T memory stem cells. Third, levels of the IP-10 cytokine directly correlated with parasite burden. These results raise fundamental questions on the priming, maintenance, and location of a B-cell population that produces high antibody levels in the face of severe B-cell deficiency. Our results should invoke interest among researchers to study the immunology and pathogenesis of human babesiosis.

Background and ObjectivesMalaria risk deferral policies are important for mitigating the risk of transfusion-transmitted malaria and apply to all transfusable components, including plasma. While donors of plasma components are deferred for malaria risk in the United States, the viability of intraerythrocytic Plasmodium falciparum parasites present in human plasma components stored under different temperatures and durations has not been previously reported.Materials and MethodsWe spiked human plasma with a low level of ring-stage P. falciparum-infected red blood cells and then determined their viability in cultures after storage at room temperature (22 degrees C), refrigeration (4 degrees C) and frozen conditions at -20 and -80 degrees C.ResultsP. falciparum parasites spiked in human plasma remained viable after storage at 22 degrees C for a maximum of 7 days. When stored at 4 degrees C, parasites were viable after 1 and 3 days of storage and only for 1 day after storage at -20 degrees C. Storage at -80 degrees C had a cryopreserving effect and parasites remained viable for up to 176 days, the longest period tested.ConclusionP. falciparum parasites can survive for short durations in human plasma when stored at room temperature, or in refrigerated or frozen conditions at -20 degrees C. However, when stored at -80 degrees C, viable parasites were detected for up to 176 days, the maximum duration for which viability was assessed. In summary, Plasmodium parasites can survive in human plasma under different storage conditions and pose a risk of transfusion-transmitted infection.

Babesia microti, an intraerythrocytic apicomplexan parasite, is the primary causative agent of human babesiosis and an emerging threat to public health in the United States and elsewhere. An effective vaccine against B. microti would reduce disease severity in acute babesiosis patients and shorten the parasitemic period in asymptomatic individuals, thereby minimizing the risk of transfusion-transmitted babesiosis. Here we report on immunogenicity, protective efficacy, and correlates of immunity following immunization with four immunodominant recombinantly produced B. microti antigens-Serine Reactive Antigen 1 (SERA1), Maltese Cross Form Related Protein 1 (MCFRP1), Piroplasm β-Strand Domain 1 (PiβS1), and Babesia microti Alpha Helical Cell Surface Protein 1 (BAHCS1)-delivered subcutaneously in Montanide ISA 51/CpG adjuvant in three doses to BALB/c mice. Following B. microti parasite challenge, BAHCS1 led to the highest reduction in peak parasitemia (67.8%), followed by SERA1 (44.8%) and MCFRP1 (41.9%); PiβS1 (27.6%) had minimal protective effect. All four B. microti antigens induced high ELISA total IgG and each isotype; however, antibody levels did not directly correlate with anti-parasitic activity in mice. Increased prechallenge levels of some cell populations including follicular helper T cells (TFH) and memory B cells, along with a set of six cytokines [IL-1α, IL-2, IL-3, IL-6, IL-12(p40), and G-CSF] that belong to both innate and adaptive immune responses, were generally associated with protective immunity. Our results indicate that mechanisms driving recombinant B. microti antigen-induced immunity are complex and multifactorial. We think that BAHCS1 warrants further evaluation in preclinical studies.

TransfusionVolume 63, Issue S5 p. 141A-142A SUPPLEMENT ARTICLE P-BB-72 | Survival of Plasmodium Falciparum Parasites in Human Plasma Under Different Storage Temperatures A. Lavrentieva, A. Lavrentieva OBRR, CBER, FDASearch for more papers by this authorM. Oakley, M. Oakley OBRR, CBER, FDASearch for more papers by this authorV. Majam, V. Majam OBRR, CBER, FDASearch for more papers by this authorA. Eder, A. Eder Office of Blood Research and Review, CBER, FDASearch for more papers by this authorC. Villa, C. Villa OBRR, CBER, FDASearch for more papers by this authorS. Kumar, S. Kumar Office of Blood Research and Review, CBER, FDASearch for more papers by this author A. Lavrentieva, A. Lavrentieva OBRR, CBER, FDASearch for more papers by this authorM. Oakley, M. Oakley OBRR, CBER, FDASearch for more papers by this authorV. Majam, V. Majam OBRR, CBER, FDASearch for more papers by this authorA. Eder, A. Eder Office of Blood Research and Review, CBER, FDASearch for more papers by this authorC. Villa, C. Villa OBRR, CBER, FDASearch for more papers by this authorS. Kumar, S. Kumar Office of Blood Research and Review, CBER, FDASearch for more papers by this author First published: 12 October 2023 https://doi.org/10.1111/trf.176_17554Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume63, IssueS5October 2023Pages 141A-142A RelatedInformation

BACKGROUND Borrelia miyamotoi is a relapsing fever spirochete that relatively recently has been reported to infect humans. It causes an acute undifferentiated febrile illness that can include meningoencephalitis and relapsing fever. Like Borrelia burgdorferi, it is transmitted by Ixodes scapularis ticks in the northeastern United States and by Ixodes pacificus ticks in the western United States. Despite reports of clinical cases from North America, Europe, and Asia, the prevalence, geographic range, and pattern of expansion of human B. miyamotoi infection are uncertain. To better understand these characteristics of B. miyamotoi in relation to other tickborne infections, we carried out a cross-sectional seroprevalence study across New England that surveyed B. miyamotoi, B. burgdorferi, and Babesia microti infections. METHODS We measured specific antibodies against B. miyamotoi, B. burgdorferi, and B. microti among individuals living in 5 New England states in 2018. RESULTS Analysis of 1153 serum samples collected at 11 catchment sites showed that the average seroprevalence for B. miyamotoi was 2.8% (range, 0.6%-5.2%), which was less than that of B. burgdorferi (11.0%; range, 6.8%-15.6%) and B. microti (10.0%; range, 6.5%-13.6%). Antibody screening within county residence in New England showed varying levels of seroprevalence for these pathogens but did not reveal a vectoral geographical pattern of distribution. CONCLUSIONS Human infections caused by B. miyamotoi, B. burgdorferi, and B. microti are widespread with varying prevalence throughout New England.

There is an urgent need for a malaria vaccine that can prevent severe disease in young children and adults. Despite earlier work showing an immunological mechanism for preventing infection and reducing disease severity, there is currently no reliable vaccine that can provide durable protection. In part, this may reflect a limited number of ways that the host can respond to the NANP repeat sequences of circumsporozoite protein (CSP) in the parasite. In addition, it may reflect antigenic escape by the parasite from protective antibodies. To be successful, a vaccine must protect against repeated exposure to infected mosquitoes in endemic areas. We have created a series of live viral vectors based on the rubella vaccine strain that express multiple tandem repeats of NANP, and we demonstrate immunogenicity in a rhesus macaque model. We tested the vectors in a sequential immunization strategy. In the first step, the animals were primed with CSP-DNA vaccine and boosted with rubella/CSP vectors. In the second step, we gave rubella/CSP vectors again, followed by recombinant CSP protein. Following the second step, antibody titers were comparable to adult exposure to malaria in an endemic area. The antibodies were specific for native CSP protein on sporozoites, and they persisted for at least 1½ years in two out of three macaques. Given the safety profile of rubella vaccine in children, these vectors could be most useful in protecting young children, who are at greatest risk of severe malarial disease.

Malaria vaccines that disrupt the Plasmodium life cycle in mosquitoes and reduce parasite transmission in endemic areas are termed transmission-blocking vaccines (TBVs). Despite decades of research, there are only a few Plasmodium falciparum antigens that indisputably and reproducibly demonstrate transmission-blocking immunity. So far, only two TBV candidates have advanced to phase 1/2 clinical testing with limited success. By applying an unbiased transcriptomics-based approach, we have identified Pf77 and male development gene 1 (PfMDV-1) as two P. falciparum TBV antigens that, upon immunization, induced antibodies that caused reductions in oocyst counts in Anopheles mosquito midguts in a standard membrane feeding assay. In-depth studies were performed to characterize the genetic diversity of, stage-specific expression by, and natural immunity to these two molecules to evaluate their suitability as TBV candidates. Pf77 and PfMDV-1 display limited antigenic polymorphism, are pan-developmentally expressed within the parasite, and induce naturally occurring antibodies in Ghanaian adults, which raises the prospect of natural boosting of vaccine-induced immune response in endemic regions. Together, these biological properties suggest that Pf77 and PfMDV-1 may warrant further investigation as TBV candidates.

Significance Novel therapies are urgently needed that can ameliorate the clinical syndromes of cerebral malaria, the most severe consequences of Plasmodium infection, and thereby reduce malaria fatality. Monoclonal antibodies that target CD47, a “don’t eat me” signal, have been demonstrated to enhance cellular clearance of cancer cells by promoting macrophage phagocytosis. We sought to adopt this therapeutic strategy to ameliorate the clinical syndromes associated with cerebral malaria with the goals of reducing disease-associated morbidity and mortality. We demonstrate that CD47 blockade by anti-CD47 injection leads to survival from cerebral malaria in mice.

Babesia spp. are tick-transmitted intra-erythrocytic protozoan parasites that infect humans and animals, causing a flu-like illness and hemolytic anemia. There is currently no human vaccine available. People most at risk of severe disease are the elderly, immunosuppressed, and asplenic individuals. B. microti and B. divergens are the predominant species affecting humans. Here, we present a whole-parasite Babesia vaccine. To establish proof-of-principle, we employed chemically attenuated B. microti parasitized red blood cells from infected mice. To aid clinical translation, we produced liposomes containing killed parasite material. Vaccination significantly reduces peak parasitemia following challenge. B cells and anti-parasite antibodies do not significantly contribute to vaccine efficacy. Protection is abrogated by the removal of CD4(+) T cells or macrophages prior to challenge. Importantly, splenectomized mice are protected by vaccination. To further facilitate translation, we prepared a culture-based liposomal vaccine and demonstrate that this performs as a universal vaccine inducing immunity against different human Babesia species.

Immunogenicity and efficacy of pre-erythrocytic malaria vaccine candidates, such as RTS,S and radiation attenuated sporozoites (RAS), has been shown to be severely compromised in clinical trials conducted in endemic regions compared to the United States. In this study, we measured the effect of prior exposure to blood stage malaria on preerythrocytic vaccine efficacy using a circumsporozoite surface protein (PyCSP) vaccine candidate in the Plasmodium yoelii 17XNL murine model of malaria. Immunization with PyCSP delivered in Montanide ISA 51 adjuvant induced antibody-dependent sterilizing immunity against sporozoite challenge in C57BL/6 mice. However, prior exposure of PyCSP vaccinated mice to blood stage malaria resulted in a significant increase in intrahepatic parasite burden after sporozoite challenge demonstrating that blood stage malaria impaired the protective efficacy of PyCSP. This loss of vaccine efficacy caused by prior exposure to blood stage malaria was accompanied by a threefold reduction in IgG1 antibody titers. Results of an ELISpot assay performed 14 weeks after vaccination indicate that prior exposure to blood stage malaria causes a significant contraction of the memory B cell response to PyCSP. This model can be used to identify novel immune regulatory pathways induced by blood stage malaria that compromise pre-erythrocytic vaccine efficacy

The development of effective malaria vaccines is hampered by incomplete understanding of the immunological correlates of protective immunity. Recently, the moderate clinical efficacy of the Plasmodium falciparum circumsporozoite protein (CSP)-based RTS,S/AS01E vaccine in phase 3 studies highlighted the urgency to design and test more efficacious next-generation malaria vaccines. In this study, we report that immunization with recombinant CSP from Plasmodium yoelii (rPyCSP), when delivered in Montanide ISA 51, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. This immunity was antibody dependent, as evidenced by the complete loss of immunity in B-cell-knockout (KO) mice and by the ability of immune sera to neutralize sporozoite infectivity in mice. Th2-type isotype IgG1 antibody levels were associated with protective immunity. The fact that immunized gamma interferon (IFN-γ)-KO mice and wild-type (WT) mice have similar levels of protective immunity and the absence of IFN-γ-producing CD4+ and CD8+ T cells in protected mice, as shown by flow cytometry, indicate that the immunity is IFN-γ independent. Protection against sporozoite challenge correlated with higher frequencies of CD4+ T cells that express interleukin-2 (IL-2), IL-4, and tumor necrosis factor alpha (TNF-α). In the RTS,S study, clinical immunity was associated with higher IgG levels and frequencies of IL-2- and TNF-α-producing CD4+ T cells. The other hallmarks of immunity in our study included an increased number of follicular B cells but a loss in follicular T helper cells. These results provide an excellent model system to evaluate the efficacy of novel adjuvants and vaccine dosage and determine the correlates of immunity in the search for superior malaria vaccine candidates.

Blood stage malaria parasites attenuated with seco‐cyclopropyl pyrrolo indole (CPI) analogues induce robust immunity in mice to homologous and heterologous malaria parasites and are being considered for the development of a human vaccine. However, it is not understood how attenuated parasites induce immunity. We showed that following vaccination, parasite DNA persisted in blood for several months, raising the possibility that ongoing immune stimulation may be critical. However, parasites were not seen microscopically beyond 24 h postvaccination. We aimed to provide a mechanistic understanding of immune induction.

Macrophages express a wide array of invariant receptors that facilitate host defense and mediate pathogenesis during pathogen invasion. We report on a novel population of CD11bhighCD14+F4/80+ macrophages that express TCRβ. This population expands dramatically during a Plasmodium berghei ANKA infection and sequesters in the brain during experimental cerebral malaria. Importantly, measurement of TCRβ transcript and protein levels in macrophages in wildtype versus nude and Rag1 knockout mice establishes that the observed expression is not a consequence of passive receptor expression due to phagocytosis or trogocytosis of peripheral T cells or nonspecific antibody staining to an Fc receptor or cross reactive epitope. We also demonstrate that TCRβ on brain sequestered macrophages undergoes productive gene rearrangements and shows preferential Vβ usage. Remarkably, there is a significant correlation in the proportion of macrophages that express TCRβ and peripheral parasitemia. In addition, presence of TCRβ on the macrophage also correlates with a significant increase (1.9 fold) in the phagocytosis of parasitized erythrocytes. By transcriptional profiling, we identify a novel set of genes and pathways that associate with TCRβ expression by the macrophage. Expansion of TCRβ-expressing macrophages points towards a convergence of the innate and adaptive immune responses where both arms of the immune system cooperate to modulate the host response to malaria and possibly other infections.