Paul R. Sleath

Broadly targeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate the memory compartments of exposed subjects

Human cytomegalovirus (HCMV) infections of immunocompetent hosts are characterized by a dynamic, life-long interaction in which host immune responses, particularly of T cells, restrain viral replication and prevent disease but do not eliminate the virus or preclude transmission. Because HCMV is among the largest and most complex of known viruses, the T cell resources committed to maintaining this balance have never been characterized completely. Here, using cytokine flow cytometry and 13,687 overlapping 15mer peptides comprising 213 HCMV open reading frames (ORFs), we found that 151 HCMV ORFs were immunogenic for CD4+ and/or CD8+ T cells, and that ORF immunogenicity was influenced only modestly by ORF expression kinetics and function. We further documented that total HCMV-specific T cell responses in seropositive subjects were enormous, comprising on average ∼10% of both the CD4+ and CD8+ memory compartments in blood, whereas cross-reactive recognition of HCMV proteins in seronegative individuals was limited to CD8+ T cells and was rare. These data provide the first glimpse of the total human T cell response to a complex infectious agent and will provide insight into the rules governing immunodominance and cross-reactivity in complex viral infections of humans.

Diversity of the CD8 + T-cell response to herpes simplex virus type 2 proteins among persons with genital herpes

ABSTRACT Cytolytic T cells play a major role in controlling herpes simplex virus type 2 (HSV-2) infections in humans. In an effort to more thoroughly evaluate the response to HSV-2 directly, ex vivo, we developed an enzyme-linked immunospot (ELISPOT) assay that utilized pools of overlapping synthetic peptides presented by autologous dendritic cells to purified CD8+ T cells. Donor response rates to individual open reading frames (ORFs) ranged from fewer than 5% responding to as many as 70% responding, with the greatest frequency of responses (by ORF) being directed against UL39, UL25, UL27, ICP0, UL46, and UL47 in descending order of frequency. HSV-2-seropositive subjects responded to as few as 3 or as many as 46 of the 48 ORFs tested, with a median of 11 ORFs recognized. HLA-B*07 expression correlated with stronger responses overall that were directed primarily against UL49 and UL46. Cumulative precursor frequencies in the blood ranged from 500 to almost 6,000 HSV-2 spot-forming units/106 CD8+ T cells. The magnitude and breadth of the response in the infected population were greater than previously appreciated. Whether this variability in the CD8+ T-cell response within individuals is associated with the frequency of viral reactivation warrants further study.

A multi-epitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in radioimmunoprecipitation-confirmed and consensus-positive sera.

Peptide epitopes of Trypanosoma cruzi have been identified through expression cloning. A tripeptide (2/D/E) containing three epitopes (TcD, TcE, PEP-2) was used in ELISA to detect antibodies to T. cruzi in 239 of 240 consensus-positive sera and 41 of 42 sera confirmed positive by radioimmunoprecipitation assay. The 1 discrepant consensus-positive serum was used to expression-clone a novel gene that contained a repeat sequence. A peptide corresponding to this sequence, TcLo1.2, was specific for T. cruzi. This antigen detected the discrepant consensus-positive serum and enhanced reactivity of low-positive sera in the tripeptide assay. A branched synthetic peptide, 2/D/E/Lo1.2, or a linear recombinant, r2/D/E/Lo1.2, realized all of the diagnostic features of the four epitopes, including the ability to boost reactivity of low-reactive sera. These studies show that peptides and recombinants containing multiple repeat epitopes are powerful tools for developing assays for T. cruzi antibody detection and have direct application in blood screening.

Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein.

ABSTRACT The interaction of the innate immune system with the microbial world involves primarily two sets of molecules generally known as microbial pattern recognition receptors and microbial pattern recognition molecules, respectively. Examples of the former are the Toll receptors present particularly in macrophages and dendritic cells. Conversely, the microbial pattern recognition molecules are conserved protist homopolymers, such as bacterial lipopolysaccharides, lipoteichoic acids, peptidoglycans, glucans, mannans, unmethylated bacterial DNA, and double-strand viral RNA. However, for protists that lack most of these molecules, such as protozoans, the innate immune system must have evolved receptors that recognize other groups of microbial molecules. Here we present evidence that a highly purified protein encoded by a Leishmania brasiliensis gene may be one such molecule. This recombinant leishmanial molecule, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), strongly stimulates spleen cells from severe combined immunodeficient (SCID) mice to produce interleukin-12 (IL-12), IL-18, and high levels of gamma interferon. In addition, LeIF potentiates the cytotoxic activity of the NK cells of these animals. Because LeIF is a conserved molecule and because SCID mice lack T and B lymphocytes but have a normal innate immune system (normal reticuloendothelial system and NK cells), these results suggest that proteins may also be included as microbial pattern recognition molecules. The nature of the receptor involved in this innate recognition is unknown. However, it is possible to exclude the Toll receptor Tlr4 as a putative LeIF receptor because the gene encoding this receptor is defective in C3H/HeJ mice, the mouse strain used in the present studies.

Identification of Babesia microti-specific immunodominant epitopes and development of a peptide EIA for detection of antibodies in serum.

BACKGROUND: Babesia microti is a tick‐borne agent that is increasingly implicated in transfusion‐acquired infection, especially in immunocompromised and elderly recipients. To develop a test that can detect antibody responses to B. microti, peptide epitopes identified in two serocomplementary B. microti‐specific antigens were used in a prototype EIA.