Discriminating Protective from Nonprotective Plasmodium-Specific CD8+ T Cell Responses.

Despite decades of research, malaria remains a global health crisis. Current subunit vaccine approaches do not provide efficient long-term, sterilizing immunity against Plasmodium infections in humans. Conversely, whole parasite vaccinations with their larger array of target Ags have conferred long-lasting sterilizing protection to humans. Similar studies in rodent models of malaria reveal that CD8(+) T cells play a critical role in liver-stage immunity after whole parasite vaccination. However, it is unknown whether all CD8(+) T cell specificities elicited by whole parasite vaccination contribute to protection, an issue of great relevance for enhanced subunit vaccination. In this article, we show that robust CD8(+) T cell responses of similar phenotype are mounted after prime-boost immunization against Plasmodium berghei glideosome-associated protein 5041-48-, sporozoite-specific protein 20318-325-, thrombospondin-related adhesion protein (TRAP) 130-138-, or circumsporozoite protein (CSP) 252-260-derived epitopes in mice, but only CSP252-260- and TRAP130-138-specific CD8(+) T cells provide sterilizing immunity and reduce liver parasite burden after sporozoite challenge. Further, CD8(+) T cells specific to sporozoite surface-expressed CSP and TRAP proteins, but not intracellular glideosome-associated protein 50 and sporozoite-specific protein 20, efficiently recognize sporozoite-infected hepatocytes in vitro. These results suggest that: 1) protection-relevant antigenic targets, regardless of their immunogenic potential, must be efficiently presented by infected hepatocytes for CD8(+) T cells to eliminate liver-stage Plasmodium infection; and 2) proteins expressed on the surface of sporozoites may be good target Ags for protective CD8(+) T cells.

Correlates of protective immunity following whole sporozoite vaccination against malaria.

Human infection with Plasmodium parasites remains a serious global health crisis, leading to more than 600,000 deaths annually. Currently, no licensed vaccine is available to alleviate this malaria disease burden and vaccination with the most advanced antimalarial vaccine candidate, RTS,S, provides limited protection that wanes over time. To date, the only vaccination strategy capable of inducing complete, long-lasting protection in human subjects is administration of attenuated whole sporozoites. Several approaches for vaccination with attenuated whole sporozoites have been clinically tested in humans and include vaccination with radiation or genetically attenuated sporozoites or with virulent sporozoites concurrent with administration of antimalarial drug cover. Rodent studies with these three attenuated whole sporozoite vaccination (WSV) approaches provide insights into the immune correlates of vaccine-induced protection. The majority of these studies have identified a critical role for liver-stage parasite-directed CD8 T cells in providing protection with possible contributions from Plasmodium-specific CD4 T cells or antibodies. Together, rodent and human vaccination studies with attenuated WSV may lead to an understanding of the correlates of protective immunity against malarial disease, and the development of new, highly efficacious vaccines.

CD8 T cell independent immunity after single dose infection-treatment-vaccination (ITV) against Plasmodium yoelii.

Sporozoite vaccination of both humans and rodents elicits potent anti-malarial immunity, but the dose of sporozoites and the number of immunizations required varies with vaccination approach. Here we examine the immunological basis for superior protection afforded from single-dose vaccination with virulent sporozoites administered under prophylatic chloroquine-cover, referred to as infection-treatment-vaccination (ITV), compared to the well-studied approach of administering radiation-attenuated Plasmodium sporozoites (RAS). Earlier rodent studies utilizing ITV and RAS vaccination suggested a major role of CD8 T cells in reducing liver parasite burden after sporozoite challenge in a BALB/c mouse model. Consistent with this, we find that in C57Bl/6 mice ITV elicits substantially higher parasite-specific CD8 T cell responses than RAS vaccination and enhances immunity against P. yoelii infection. However, we show ITV-induced CD8 T cells are not necessary for protection following liver-stage sporozoite or blood-stage parasite challenge. Mechanistically, we found protection afforded from single-dose ITV is associated with low grade, transient parasitemia shortly following cessation of chloroquine treatment and generation of potent antibody responses to blood-stage parasites. Collectively, our data show the mechanistic basis for enhanced protective immunity against P. yoelli elicited by ITV in highly susceptible C57Bl/6 mice is independent of CD8 T cells. These studies may be relevant in understanding the potent immunity observed with ITV in humans.

Tracking the total CD8 T cell response following whole Plasmodium vaccination.

CD8 T cells are critical mediators of protection against Plasmodium liver-stage infection. Most studies of the CD8 T cell response to whole parasite Plasmodium vaccines address a single T cell epitope in BALB/c mice, and thus provide limited information. Here, we describe a surrogate activation marker approach that uses the coordinate downregulation of the CD8α chain and upregulation of the integrin CD11a to track the total CD8 T cell response to Plasmodium vaccination via flow cytometry. With this approach, quantitative (magnitude, kinetics) and qualitative (distribution, phenotype, and function) features of the total CD8 T cell response to vaccination with attenuated Plasmodium or other pathogens can be studied.

Identification of single nucleotide polymorphisms associated with hyperproduction of alpha-toxin in Staphylococcus aureus.

The virulence factor α-toxin (hla) is needed by Staphylococcus aureus in order to cause infections in both animals and humans. Although the complicated regulation of hla expression has been well studied in human S. aureus isolates, the mechanisms of of hla regulation in bovine S. aureus isolates remain undefined. In this study, we found that many bovine S. aureus isolates, including the RF122 strain, generate dramatic amounts of α-toxin in vitro compared with human clinical S. aureus isolates, including MRSA WCUH29 and MRSA USA300. To elucidate potential regulatory mechanisms, we analyzed the hla promoter regions and identified predominant single nucleotide polymorphisms (SNPs) at positions -376, -483, and -484 from the start codon in α-toxin hyper-producing isolates. Using site-directed mutagenesis and hla promoter-gfp-luxABCDE dual reporter approaches, we demonstrated that the SNPs contribute to the differential control of hla expression among bovine and human S. aureus isolates. Using a DNA affinity assay, gel-shift assays and a null mutant, we identified and revealed that an hla positive regulator, SarZ, contributes to the involvement of the SNPs in mediating hla expression. In addition, we found that the bovine S. aureus isolate RF122 exhibits higher transcription levels of hla positive regulators, including agrA, saeR, arlR and sarZ, but a lower expression level of hla repressor rot compared to the human S. aureus isolate WCUH29. Our results indicate α-toxin hyperproduction in bovine S. aureus is a multifactorial process, influenced at both the genomic and transcriptional levels. Moreover, the identification of predominant SNPs in the hla promoter region may provide a novel method for genotyping the S. aureus isolates.

A community study of language concordance in Russian patients with diabetes.

PURPOSE: Unique challenges exist in the provision of culturally and linguistically appropriate health care to ethnically diverse populations. The purpose of this study was to improve the metabolic control of Russian patients with diabetes through increased attention to language and cultural concordance between the provider and patient. PROCEDURES: This is a retrospective cohort study of 55 Russian immigrant type 2 patients with diabetes to assess levels of glycemic, lipid, and blood pressure control before and after the arrival of a bilingual Russian internist trained in both the United States and Russia. Paired t test was used to compare these parameters before and after the bilingual Russian provider joined our practice. FINDINGS: Overall, the mean low density lipoprotein (LDL) level decreased by 20% from 126 mg/dL to 102 mg/dL (P=.0002) respectively before and after the Russian provider began treating these patients. Mean hemoglobin A1c decreased from 8.4% to 8.0% (P=.007), and diastolic blood pressure was reduced from 82.7 mm Hg to 76.3 mm Hg (P=.0002). Systolic blood pressure also improved from 143.2 mm Hg to 140.6 mm Hg (P=.3). At the end of the study period more than 90% of the patients with diabetes were appropriately taking lipid-lowering medications and an angiotensin converting enzyme (ACE) inhibitor. CONCLUSION: Our findings suggest that it is important to facilitate the health care of ethnically diverse minority populations in a manner that attempts to maximize language and cultural concordance. These potential benefits will assume an even greater importance with the expansive growth of ethnic minorities in the United States and their unique healthcare needs.