Influence of HCV genotype and co-infection with human immunodeficiency virus on CD4(+) and CD8(+) T-cell responses to hepatitis C virus

The role of T-cells in clearance of hepatitis C virus (HCV) during acute infection is critical. The relevance of the immunological response in the control of HCV replication is less clear in chronic HCV infection. HCV-specific T-cell responses were examined in 92 interferon-naive individuals with chronic hepatitis C. A panel of 441 overlapping peptides spanning all expressed HCV proteins was used to measure HCV-specific T-cell responses, using flow cytometry after stimulating peripheral blood mononuclear cells (PBMCs) with different pools of these peptides. Most patients showed responses to at least one HCV protein, with NS5B for CD8(+) responses and E2 for CD4(+) responses identified most frequently. Both the prevalence and breadth of CD4(+) and CD8(+) responses were lower in co-infected patients, independently of the HCV genotype.     

CD4+CD25+Foxp3+IFN-γ+ human induced T regulatory cells are induced by interferon-γ and suppress alloresponses nonspecifically

Interferon-γ (IFN-γ)-producing CD3(+)CD4(+)CD25(+)Foxp3(+) peripheral blood lymphocytes (PBL) are more frequently detectable in patients with good than in patients with impaired long-term kidney graft function, suggesting an immunoregulatory role of this induced T regulatory (iTreg) subtype. Herein, the in vitro function of separated CD3(+)CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL that were induced by phorbol 12-myristate 13-acetate (PMA)/ionomycin or alloantigenic stimulation was investigated using cell coculture techniques and flow cytometry. CD4(+)CD25(+)Foxp3(+) PBL with intracellular IFN-γ production increased to 26% in cell cultures stimulated with PMA/ionomycin for 6 hours. Recombinant IFN-γ augmented and anti-IFN-γ monoclonal antibody blocked induction of CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL, suggesting their IFN-γ-dependent induction. In addition, CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL produced immunosuppressive interleukin (IL)-10, transforming growth factor-β, and IL-4 intracellularly and expressed both IFN-γ and IFN-γ receptors (CD119) on the cell surface, allowing separation of CD4(+)CD25(+)IFN-γ(+) PBL with 98% purity. Addition of enriched CD4(+)CD25(+)IFN-γ(+) PBL to autologous PMA/ionomycin stimulated PBL decreased blast formation (p < 0.05), indicating suppression of cell proliferation by CD4(+)CD25(+)IFN-γ(+) PBL. CD4(+)CD25(+)IFN-γ(+) PBL separated from primary mixed leukocyte cultures (MLC) and added to autologous or third-party secondary MLC suppressed allogeneic T-cell activation nonspecifically (p < 0.05). We conclude that CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL are induced by IFN-γ, making them sensors for IFN-γ and initial immune responses. Circulating CD4(+)CD25(+)Foxp3(+)IFN-γ(+) PBL could suppress allogeneic T-cell responses in patients and may be involved in inhibition of the posttransplant alloresponse.     

Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients

The role of cell-mediated immunity in human SARS-CoV infection is still not well understood. In this study, we found that memory T-cell responses against the spike (S) protein were persistent for more than 1 year after SARS-CoV infection by detecting the production of IFN-gamma using ELISA and ELISpot assays. Flow cytometric analysis showed that both CD4(+) and CD8(+) T cells were involved in cellular responses against SARS-CoV infection. Interestingly, most of SARS-CoV S-specific memory CD4(+) T cells were central memory cells expressing CD45RO(+) CCR7(+) CD62L(-). However, the majority of memory CD8(+) T cells revealed effector memory phenotype expressing CD45RO(-) CCR7(-) CD62L(-). Thus, our study provides the evidence that SARS-CoV infection in humans can induce cellular immune response that is persistent for a long period of time. These data may have an important implication in the possibility of designing effective vaccine against SARS-CoV infection, specifically in defining T-cell populations that are implicated in protective immunity.     

Genome-wide screening of human T-cell epitopes in influenza A virus reveals a broad spectrum of CD4 T-cell responses to internal proteins, hemagglutinins, and neuraminidases

We performed a genome-wide screening for T-cell epitopes using synthetic peptides that encompass all of the influenza A viral proteins, including subtype variants for hemagglutinin (HA; H1, H3, and H5) and neuraminidase (NA; human and avian N1 and N2) proteins, based on the sequence information of recently circulating strains. We identified a total of 83 peptides, 54 of them novel, to which specific T cells were detectable in interferon-γ (IFN-γ) enzyme-linked immunosorbent spot assays using peripheral blood mononuclear cells from four healthy adult donors. The surface glycoproteins, HA and NA, major components of vaccines, expressed many T-cell epitopes. HA and matrix protein 1 expressed more T-cell epitopes than other viral proteins, most of which were recognized by CD4⁺ T cells. We established several cytotoxic CD4⁺ T-cell lines from these donors. We also analyzed H1 and H3 HA-specific T-cell responses using the peripheral blood mononuclear cells of 30 hospital workers. Fifty-three percent of donors gave a positive response to H3 HA peptides, whereas 17% gave a positive response to H1 HA peptides. Our genome-wide screening is useful in identifying T-cell epitopes and is complementary to the approach based on the predicted binding peptides to well-studied HLA-A, -B, and -DR alleles.     

Identification of a Promiscuous T-Cell Epitope in Mycobacterium tuberculosis Mce Proteins

The characterization of Mycobacterium tuberculosis antigens inducing CD4(+) T-cell responses could critically contribute to the development of subunit vaccines for M. tuberculosis. Here we performed computational analysis by using T-cell epitope prediction software (known as TEPITOPE) to predict promiscuous HLA-DR ligands in the products of the mce genes of M. tuberculosis. The analysis of the proliferative responses of CD4(+) T cells from patients with pulmonary tuberculosis to selected peptides displaying promiscuous binding to HLA-DR in vitro led us to the identification of a peptide that induced proliferation of CD4(+) cells from 50% of the tested subjects. This study demonstrates that a systematic computational approach can be used to identify T-cell epitopes in proteins expressed by an intracellular pathogen.     

The involvement of natural killer cells in the pathogenesis of severe acute respiratory syndrome

By using peripheral blood samples from 221 cases of severe acute respiratory syndrome (SARS), 34 of Mycoplasma pneumoniae infection, and 44 healthy adults, we measured the total number of natural killer (NK) and CD158b+ NK cells (CD158b+ NK) using flow cytometric analysis and calculated the percentage of CD158b+ NK cells. The total number of NK and CD158b+ NK cells and the percentage of CD158b+ NK cells were significantly lower in patients with SARS than in those with M. pneumoniae infection (P < .05 for all) and healthy subjects (P < .01, P < .01, P < .05, respectively); in 72 patients with severe SARS than in 149 with mild SARS (P < .05 for all); and in 174 cases of SARS with anti-SARS coronavirus-specific IgG and/or IgM antibodies than in 47 without antibodies (P < .05, P < .01, P < .01, respectively). There were no significant differences for the 3 values among patients with SARS without anti-SARS coronavirus antibody, patients with M. pneumoniae infection, and healthy subjects. The number of NK cells and the expression of CD158b on the surface of NK cells changed in patients with SARS and correlated with disease severity and the presence of anti-SARS coronavirus-specific antibodies; SARS differed from M. pneumoniae infection in pathogenesis involving NK cells. Monitoring the total number of NK and CD158b+ NK cells and the percentage of CDD158b+ NK cells might aid in differentiating SARS from M. pneumoniae infection.     

CD4+ T-Cell- and Gamma Interferon-Dependent Protection against Murine Malaria by Immunization with Linear Synthetic Peptides from a Plasmodium yoelii 17-Kilodalton Hepatocyte Erythrocyte Protein

Most work on protective immunity against the pre-erythrocytic stages of malaria has focused on induction of antibodies that prevent sporozoite invasion of hepatocytes, and CD8(+) T-cell responses that eliminate infected hepatocytes. We recently reported that immunization of A/J mice with an 18-amino-acid synthetic linear peptide from Plasmodium yoelii sporozoite surface protein 2 (SSP2) in TiterMax adjuvant induces sterile protection that is dependent on CD4(+) T cells and gamma interferon (IFN-gamma). We now report that immunization of inbred A/J mice and outbred CD1 mice with each of two linear synthetic peptides from the 17-kDa P. yoelii hepatocyte erythrocyte protein (HEP17) in the same adjuvant also induces protection against sporozoite challenge that is dependent on CD4(+) T cells and IFN-gamma. The SSP2 peptide and the two HEP17 peptides are recognized by B cells as well as T cells, and the protection induced by these peptides appears to be directed against the infected hepatocytes. In contrast to the peptide-induced protection, immunization of eight different strains of mice with radiation-attenuated sporozoites induces protection that is absolutely dependent on CD8(+) T cells. Data represented here demonstrate that CD4(+) T-cell-dependent protection can be induced by immunization with linear synthetic peptides. These studies therefore provide the foundation for an approach to pre-erythrocytic-stage malaria vaccine development, based on the induction of protective CD4(+) T-cell responses, which will complement efforts to induce protective antibody and CD8(+) T-cell responses.     

Identification of MHC class II restricted T-cell-mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides

Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide-based vaccines. In the present work, bioinformatics technology was employed to predict binding motifs of 9mer peptides derived from M. tuberculosis for the 12 HLA-I supertypes. Subsequently, the predicted peptides were synthesized and assayed for binding to HLA-I molecules in a biochemically based system. The antigenicity of a total of 157 peptides with measured affinity for HLA-I molecules of K(D) ≤ 500 nM were evaluated using peripheral blood T cells from strongly purified protein derivative reactive healthy donors. Of the 157 peptides, eight peptides (5%) were found to induce T-cell responses. As judged from blocking with HLA class I and II subtype antibodies in the ELISPOT assay culture, none of the eight antigenic peptides induced HLA class I restricted CD8(+) T-cell responses. Instead all responses were blocked by pan-HLA class II and anti-HLA-DR antibodies. In addition, CD4(+) T-cell depletion before the 10 days of expansion, resulted in total loss of reactivity in the ELISPOT culture for most peptide specificities. FACS analyses with intracellular interferon-γ staining of T cells expanded in the presence of M. tuberculosis peptides confirmed that the responsive cells were indeed CD4(+). In conclusion, T-cell immunity against HLA-I binding 9mer M. tuberculosis-derived peptides might in many cases turn out to be mediated by CD4(+) T cells and restricted by HLA-II molecules. The use of 9mer peptides recognized by both CD8(+) and CD4(+) T cells might be of importance for the development of future M. tuberculosis peptide-based vaccines.     

Identification of murine CD8 T cell epitopes in codon-optimized SARS-associated coronavirus spike protein

The causative agent of severe acute respiratory syndrome (SARS) has been identified as a new type of coronavirus, SARS-associated coronavirus (SARS-CoV). CD8 T cells play an important role in controlling diseases caused by other coronaviruses and in mediating vaccine-induced protective immunity in corresponding animal models. The spike protein, a main surface antigen of SARS-CoV, is one of the most important antigen candidates for vaccine design. Overlapping peptides were used to identify major histocompatibility complex class I-restricted epitopes in mice immunized with vectors encoding codon-optimized SARS-CoV spike protein. CD8 T-cell responses were mapped to two H-2(b)-restricted epitopes (S436-443 and S525-532) and one H-2(d)-restricted epitope (S366-374). The identification of these epitopes will facilitate the evaluation of vaccine strategies in murine models of SARS-CoV infection. Furthermore, codon and promoter optimizations can greatly enhance the overall immunogenicity of spike protein in the context of replication-defective human and simian adenoviral vaccine carriers. The optimized recombinant adenoviral vaccine vectors encoding spike can generate robust antigen-specific cellular immunity in mice and may potentially be useful for control of SARS-CoV infection.     

Development of a whole blood intracellular cytokine staining assay for mapping CD4(+) and CD8(+) T-cell responses across the HIV-1 genome

A whole blood peptide mapping intracellular cytokine staining (ICS) assay was developed that allows the direct comparison, at the individual peptide level, of CD4(+) and CD8(+) T-cell responses that span every encoded protein, in patients infected with HIV-1. Whole blood samples from HIV-1 infected patients were stimulated with overlapping synthetic peptides spanning nine subtype C HIV-1 gene regions (Gag, Pol, Nef, Env, Tat, Rev, Vif, Vpu, Vpr). Following stimulation and permeabilization, cells were stained with fluorochrome labelled antibodies to CD3, CD8 (CD4(+) cells were defined as CD8 negative cells), and IL-2 and IFN-gamma. A total of 396 overlapping peptides were arranged in pools with a matrix design which allowed the identification of individual peptide responses from multiple pool responses. HIV-1 infected patients screened using this method showed a broad range of peptide responses across the entire HIV-1 genome with CD8 T-cell responses being higher in frequency in magnitude than CD4(+) T-cell responses. The advantages of this whole blood ICS assay include the following: (1) the response to all potential HIV-1 epitopes across the genome can be examined, (2) the responding cell type can be monitored in the same reaction, and (3) considerably less blood is required than would be necessary if peripheral blood mononuclear cells (PBMC) were first isolated prior to peptide stimulation.     

Schistosoma japonicum egg antigens stimulate CD4 CD25 T cells and modulate airway inflammation in a murine model of asthma

A number of epidemiological and clinical studies have suggested an inverse association between allergy and helminth infection, such as Schistosomiasis. Therefore, we hypothesize that Schistosoma japonicum egg antigens, a type of native antigen, can induce production of CD4(+) CD25(+) T cells with regulatory activity, modulating airway inflammation and inhibiting asthma development. The frequency of CD4(+) CD25(+) T cells was determined by flow cytometry for mice treated with ovalbumin (OVA), CD25(+) depletion/OVA, schistosome egg antigens, schistosome egg antigens/OVA and for control mice. The ability of CD25(+) T cells from these mice to suppress T-cell proliferation and cytokine production was investigated both in vivo and in vitro. Results showed that the CD4(+) CD25(+) T cells of OVA-treated mice exhibited impaired control of dysregulated mucosal T helper 2 responses compared to the controls (P < 0.05). Depletion of CD25(+) cells accelerated OVA-induced airway inflammation and increased the expression of interleukin (IL)-5 and IL-4. Treatment with schistosome egg antigens increased the number and suppressive activity of CD4(+) CD25(+) T cells, which made IL-10, but little IL-4. In a murine model of asthma, S. japonicum egg antigens decreased the expression of Th2 cytokines, relieved antigen-induced airway inflammation, and inhibited asthma development. Thus, we provided evidence that S. japonicum egg antigens induced the production of CD4(+) CD25(+) T cells, resulting in constitutive immunosuppressive activity and inhibition of asthma development. These results reveal a novel form of protection against asthma and suggest a mechanistic explanation for the protective effect of helminth infection on the development of allergy.     

SARS CoV-2 infections in healthcare workers with a pre-existing T-cell response: a prospective cohort study

ObjectiveT-cell responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are observed in unexposed individuals. We evaluated the impact of this pre-existing cellular response on incident SARS-CoV-2 infections.MethodsThis was a follow-up study of 38 seronegative healthcare workers (HCWs) with previous evaluation of CD8+ and CD4+ T-cell responses after stimulation with SARS-CoV-2 structural proteins. Infection was considered in the presence of a positive RT-PCR test and/or confirmed seroconversion.ResultsTwenty of the 38 HCWs included (53%) had a previous specific CD8+ T-cell response to peptides encompassing the spike protein (S) in 13 (34%), the membrane (M) in 17 (45%), or/and the nucleocapsid (N) in three (8%). During a follow-up of 189 days (interquartile range (IQR) 172–195), 11 HCWs (29%) had an RT-PCR-positive test (n = 9) or seroconverted (n = 2). Median duration of symptoms was 2 days (IQR 0–7), and time to negative RT-PCR was 9 days (IQR 4–10). Notably, six incident infections (55%) occurred in HCWs with a pre-existing T-cell response (30% of those with a cellular response), who showed a significantly lower duration of symptoms (three were asymptomatic). Three of the six HCWs having a previous T-cell response continued to test seronegative. All the infected patients developed a robust T-cell response to different structural SARS-CoV-2 proteins, especially to protein S (91%).ConclusionA pre-existing T-cell response does not seem to reduce incident SARS-CoV-2 infections, but it may contribute to asymptomatic or mild disease, rapid viral clearance and differences in seroconversion.     

SARS coronavirus nucleocapsid immunodominant T-cell epitope cluster is common to both exogenous recombinant and endogenous DNA-encoded immunogens

Correspondence between the T-cell epitope responses of vaccine immunogens and those of pathogen antigens is critical to vaccine efficacy. In the present study, we analyzed the spectrum of immune responses of mice to three different forms of the SARS coronavirus nucleocapsid (N): (1) exogenous recombinant protein (N-GST) with Freund's adjuvant; (2) DNA encoding unmodified N as an endogenous cytoplasmic protein (pN); and (3) DNA encoding N as a LAMP-1 chimera targeted to the lysosomal MHC II compartment (p-LAMP-N). Lysosomal trafficking of the LAMP/N chimera in transfected cells was documented by both confocal and immunoelectron microscopy. The responses of the immunized mice differed markedly. The strongest T-cell IFN-gamma and CTL responses were to the LAMP-N chimera followed by the pN immunogen. In contrast, N-GST elicited strong T cell IL-4 but minimal IFN-gamma responses and a much greater antibody response. Despite these differences, however, the immunodominant T-cell ELISpot responses to each of the three immunogens were elicited by the same N peptides, with the greatest responses being generated by a cluster of five overlapping peptides, N76-114, each of which contained nonameric H2d binding domains with high binding scores for both class I and, except for N76-93, class II alleles. These results demonstrate that processing and presentation of N, whether exogenously or endogenously derived, resulted in common immunodominant epitopes, supporting the usefulness of modified antigen delivery and trafficking forms and, in particular, LAMP chimeras as vaccine candidates. Nevertheless, the profiles of T-cell responses were distinctly different. The pronounced Th-2 and humoral response to N protein plus adjuvant are in contrast to the balanced IFN-gamma and IL-4 responses and strong memory CTL responses to the LAMP-N chimera.     

Immunological responses against SARS-coronavirus infection in humans

Since the outbreak of a SARS epidemic last year, significant advances have been made on our understanding of the mechanisms of interaction between the SARS coronavirus (CoV) and the immune system. Strong humoral responses have been found in most patients following SARS-CoV infection, with high titers of neutralizing Abs present in their convalescent sera. The nucleocapsid (N) and spike (S) proteins of SARS-CoV appear to be the dominant antigens recognized by serum Abs. CD4+ T cell responses against the N protein have been observed in SARS patients and an HLA-A2-restricted cytotoxic T lymphocyte epitope in the S protein has been identified. It is likely that the immune responses induced by SARS-CoV infection could also cause pathological damage to the host, especially in the case of proinflammatory cytokines. There is also evidence suggesting that SARS-CoV might be able to directly invade cells of the immune system. Our understanding on the interaction between SARS-CoV, the immune system and local tissues is essential to future diagnosis, control and treatment of this very contagious disease.     

CD4(+) T-cell responses to bovine viral diarrhoea virus in cattle

Friesian calves were infected with one of three isolates of bovine viral diarrhoea virus (BVDV) and used to establish parameters for an in vitro model of BVDV-reactive T-cell responses in cattle. The study assessed virus clearance, seroconversion, maturation of lymphoproliferative responses (both during and following disease resolution) and the antigen-specificity of CD4(+) T cells from recovered animals. Seroconversion and virus-specific lymphoproliferation were not detected until viraemia had resolved. Interestingly, lymphoproliferation was detected earlier in the animals infected with cytopathic viruses than in those infected with noncytopathic virus despite broadly similar rates of virus clearance and seroconversion for both biotypes. CD4(+) and CD8(+) T cells were induced to proliferate by virus-infected stimulator cells whereas only CD4(+) T cells responded to non-infectious antigens. Lymphoproliferation was strain cross-reactive and MHC-restricted. Induction of T-cell proliferation by recombinant proteins identified the major envelope proteins E(rns) and E2 and the nonstructural (NS) 2-3 protein as T-cell determinants. In addition, the capsid (C) and/or the amino-terminal proteinase, N(pro) were identified as T-cell determinants from the responses of short-term T-cell lines. Thus, in this model, the CD4(+) T-cell repertoire induce by acute BVDV infection includes at least the major envelope proteins, NS2-3, and capsid and/or N(pro).     

Properties of CD4(+) T cells in human cytomegalovirus infection

The correlates of protective immunity to disease-inducing viruses in man remain to be elucidated. We determined the kinetics and properties of cytomegalovirus (CMV)-specific CD4(+) T cells in healthy individuals and renal transplant recipients during different stages of CMV infection. Our data reveal that circulating CMV-specific CD4(+) T cells displayed an effector-memory phenotype, and produced the T helper 1 cytokines interferon-gamma and tumor necrosis factor-alpha. In addition, they lacked molecules for secondary lymphoid organ homing and expressed the cytotoxic molecule granzyme B, inferring a direct role of these cells at target sites of infection. In asymptomatic individuals the CMV-specific CD4(+) T-cell response preceded CMV-specific CD8(+) T-cell responses, whereas in symptomatic individuals the CMV-specific effector memory CD4(+) T-cell response was delayed and only detectable after antiviral therapy. The appearance of disease symptoms in these patients suggests that functional CD8(+) T cell and antibody responses are insufficient to control viral replication and that formation of effector memory CD4(+) T cells is necessary for recovery of infection.     

Type I interferon supports primary CD8+ T-cell responses to peptide-pulsed dendritic cells in the absence of CD4+ T-cell help

CD8(+) T-cell responses to non-pathogen, cell-associated antigens such as minor alloantigens or peptide-pulsed dendritic cells (DC) are usually strongly dependent on help from CD4(+) T cells. However, some studies have described help-independent primary CD8(+) T-cell responses to cell-associated antigens, using immunization strategies likely to trigger natural killer (NK) cell activation and inflammatory cytokine production. We asked whether NK cell activation by MHC I-deficient cells, or administration of inflammatory cytokines, could support CD4(+) T-cell help-independent primary responses to peptide-pulsed DC. Injection of MHC I-deficient cells cross-primed CD8(+) T-cell responses to the protein antigen ovalbumin (OVA) and the male antigen HY, but did not stimulate CD8(+) T-cell responses in CD4-depleted mice; hence NK cell stimulation by MHC I-deficient cells did not replace CD4(+) T-cell help in our experiments. Dendritic cells cultured with tumour necrosis factor-α (TNF-α) or type I interferon-α (IFN-α) also failed to prime CD8(+) T-cell responses in the absence of help. Injection of TNF-α increased lymph node cellularity, but did not generate help-independent CD8(+) T-cell responses. In contrast, CD4-depleted mice injected with IFN-α made substantial primary CD8(+) T-cell responses to peptide-pulsed DC. Mice deficient for the type I IFN receptor (IFNR1) made CD8(+) T-cell responses to IFNR1-deficient, peptide-pulsed DC; hence IFN-α does not appear to be a downstream mediator of CD4(+) T-cell help. We suggest that primary CD8(+) T-cell responses will become help-independent whenever endogenous IFN-α secretion is stimulated by tissue damage, infection, or autoimmune disease.     

Major histocompatibility complex class II DR-restricted memory CD4(+) T lymphocytes recognize conserved immunodominant epitopes of Anaplasma marginale major surface protein 1a

Native major surface protein 1 (MSP1) of Anaplasma marginale, composed of covalently associated MSP1a and MSP1b proteins, stimulates protective immunity in cattle against homologous and heterologous strain challenge. Protective immunity against pathogens in the family Anaplasmataceae involves both CD4(+) T cells and neutralizing immunoglobulin G. Thus, an effective vaccine should contain both CD4(+) T- and B-lymphocyte epitopes that will elicit strong memory responses upon infection with homologous and heterologous strains. Previous studies demonstrated that the predominant CD4(+) T-cell response in MSP1 vaccinates is directed against the MSP1a subunit. The present study was designed to identify conserved CD4(+) T-cell epitopes in MSP1a presented by a broadly represented subset of major histocompatibility complex (MHC) class II molecules that would be suitable for inclusion in a recombinant vaccine. Transmembrane protein prediction analysis of MSP1a from the Virginia strain revealed a large hydrophilic domain (HD), extending from amino acids (aa) 1 to 366, and a hydrophobic region extending from aa 367 to 593. The N terminus (aa 1 to 67) includes one 28-aa form A repeat and one 29-aa form B repeat, which each contain an antibody neutralization-sensitive epitope [Q(E)ASTSS]. In MSP1 vaccinates, recombinant MSP1a HD (aa 1 to 366) stimulated recall proliferative responses that were comparable to those against whole MSP1a excluding the repeat region (aa 68 to 593). Peptide mapping determined a minimum of five conserved epitopes in aa 151 to 359 that stimulated CD4(+) T cells from cattle expressing DR-DQ haplotypes common in Holstein-Friesian breeds. Peptides representing three epitopes (aa 231 to 266, aa 270 to 279, and aa 290 to 319) were stimulatory for CD4(+) T-cell clones and restricted by DR. A DQ-restricted CD4(+) T-cell epitope, present in the N-terminal form B repeat (VSSQSDQASTSSQLG), was also mapped using T-cell clones from one vaccinate. Although form B repeat-specific T cells did not recognize the form A repeat peptide (VSSQS_EASTSSQLG), induction of T-cell anergy by this peptide was ruled out. The presence of multiple CD4(+) T-cell epitopes in the MSP1a HD, in addition to the neutralization-sensitive epitope, supports the testing of this immunogen for induction of protective immunity against A. marginale challenge.     

Virus-specific T-cell responses associated with hepatitis C virus (HCV) persistence in the liver after apparent recovery from HCV infection

Hepatitis C virus (HCV) RNA persistence in the liver has been described even after apparent resolution of HCV infection. Because T-cell reactivity plays a role in recovery from HCV infection, virus-specific T-cell responses were investigated in apparently recovered individuals in whom hepatic HCV RNA persistence was documented: 15 sustained virological responders to interferon (IFN)-treatment and 9 asymptomatic aviremic anti-HCV carriers. HCV-specific CD4(+) T-cell proliferative responses were detected significantly more often in apparently recovered individuals (sustained virological responders: 60%; asymptomatic anti-HCV carriers: 66%) compared with 50 chronic hepatitis C patients (28%; P < 0.05). However, T-cell frequencies and numbers tended to decline over time and the number of HCV proteins targeted by CD4(+) T-cell proliferative responses was limited. Interestingly, liver viral load correlated inversely with virus-specific immune responses. Thus, CD4(+) T-cell responders showed significantly lower hepatic HCV RNA levels (P < 0.05). HCV-specific IFN-gamma-secreting CD4(+) T-cells were not detected in all the apparently recovered patients although they were found significantly more often compared with chronic hepatitis C patients (P < 0.05). Also, HCV NS3-specific CD8(+) T-cells were detected in 11 HLA-A2-positive apparently recovered individuals (8 sustained virological responders and 3 asymptomatic anti-HCV carriers); T-cell frequencies tended to be greater in those patients who had lower hepatic viral levels. In conclusion, HCV-specific T-cells are detectable in apparently recovered individuals in whom HCV RNA can persist in the liver indicating that HCV replication may be prolonged in the face of an insufficient or inadequate virus-specific CD4(+) and CD8(+) T-cell response.