Vaccination in humans generates broad T cell cytokine responses

In recent years, the quantification of T cell responses to pathogens or immunogens has become a common tool in the evaluation of disease pathogenesis or vaccine immunogenicity. Such measurements are usually limited to enumerating IFN-gamma-producing cells after ex vivo stimulation with Ag, but little is known about the phenotype or complete functional repertoire of the Ag-specific cells. We used 12-color flow cytometry to characterize Ag-specific T cells elicited by vaccines or natural infection to determine lineage and differentiation status as well as the capacity to produce four cytokines (IFN-gamma, TNF-alpha, IL-2, and IL-4) and a chemokine (MIP1beta). As expected, responding cells had a typical memory phenotype; however, the cytokine profiles associated with the responses were highly complex. The pattern of cytokine coexpression in response to specific Ags was a skewed subset of the complete repertoire (revealed by polyclonal stimulation). We found significant differences in the patterns of cytokines elicited by vaccination (where IFN-gamma was by far a subdominant response) vs natural infection; in addition, there was fairly significant intersubject variation. Our findings illustrate the limitation of the evaluation of immune responses using single functional measurements (such as IFN-gamma); in fact, it is likely that sensitive evaluation of Ag-specific T cells will require the coordinate measurement of several cytokines. The presence and variability of these complex response profiles introduce the possibility that selective functional expression patterns may provide correlates for vaccine efficacy or disease progression.     

Antibody responses to individual proteins of SARS coronavirus and their neutralization activities

A novel coronavirus, the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), was identified as the causative agent of SARS. The profile of specific antibodies to individual proteins of the virus is critical to the development of vaccine and diagnostic tools. In this study, 13 recombinant proteins associated with four structural proteins (S, E, M and N) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b) of the SARS-CoV were prepared and used for screening and monitoring their specific IgG antibodies in SARS patient sera by protein microarray. Antibodies to proteins S, 3a, N and 9b were detected in the sera from convalescent-phase SARS patients, whereas those to proteins E, M, 3b, 6 and 7a were undetected. In the detectable specific antibodies, anti-S and anti-N were dominant and could persist in the sera of SARS patients until week 30. Among the rabbit antisera to recombinant proteins S3, N, 3a and 9b, only anti-S3 serum showed significant neutralizing activity to the SARS-CoV infection in Vero E6 cells. The results suggest (1) that anti-S and anti-N antibodies are diagnostic markers and in particular that S3 is immunogenic and therefore is a good candidate as a subunit vaccine antigen; and (2) that, from a virus structure viewpoint, the presence in some human sera of antibodies reacting with two recombinant polypeptides, 3a and 9b, supports the hypothesis that they are synthesized during the virus cycle.     

Immune responses and histopathological changes in rabbits immunized with inactivated SARS coronavirus

To evaluate the immunogenicity of inactivated SARS coronavirus (SARS-CoV), three groups of rabbits were immunized three times at 2-week intervals with inactivated vaccine + adjuvant, adjuvant, and normal saline respectively. Eight batchs of serum were sampled from the auricular vein at day 7 to day 51, and specific IgG antibody titers and neutralizing antibody titers were detected by indirect ELISA and micro-cytopathic effect neutralizing test. Antibody specificity was identified by proteinchip assay. Histopathological changes were detected by H&E staining. The results showed that, rabbits in the experimental group immunized with inactivated SARS-CoV all generated specific IgG antibodies with neutralizing activity, which suggested the inactivated SARS-CoV could preserve its antigenicity well and elicit an effective humoral immune responses. The peak titer value of specific IgG antibody and neutralizing antibody reached 1:40960 and 1:2560 respectively. In the experimental group, no obvious histopathological changes was detected in the H&E stained slides of heart, spleen, kidney and testis samples, but the livers had slight histopathological changes, and the lungs presented remarkable histopathological changes. These findings are of importance for SARS-CoV inactivated vaccine development. Foundation item: Joint funds of National Natural Science Foundation of China (U0632010); Program of Guangdong Provincial Key Lab of Bioengineering Medicine (51207026).     

SARS病毒:非典型肺炎相关病毒

SARS是目前在世界范围内流行的严重呼吸系统疾病。SARS病毒是有包膜的正链RNA病毒,属冠状病毒科,为新近分离鉴定的该疾病相关病原体。初步预测该病毒的复制周期与其他冠状病毒类似,但其细胞膜受体结合蛋白S的S1区、M跨膜糖蛋白等部分则存在较大的变异,可能是该病毒发生宿主改变的原因之一。此外,对SARS病毒的检测、临床诊断等方面也取得了一定的进展。     

T cell responses to Chlamydia trachomatis

Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States, as well as the leading cause of preventable blindness worldwide. Immunity to C. trachomatis requires a variety of cell types, each employing an array of effector functions. Recent work has demonstrated that both CD4+ and CD8+ T lymphocytes play a major role in protective immunity to C. trachomatis, predominantly through their secretion of interferon-gamma. This review describes the generation of acquired immunity to C. trachomatis and focuses on how T cells contribute to both protection and immunopathology.     

T cell responses to Listeria monocytogenes

Owing to its unique intracellular biology that allows it to gain access to the host cell cytosol, Listeria monocytogenes induces potent, protective CD8 responses. The study of these responses has served as a paradigm to understand cell-mediated immunity to microbial pathogens. The availability of mutants specifically defective in unique aspects of the intracellular biology of this pathogen has greatly aided these studies. During the past few years, progress has been made to understand the contribution of the innate immune system and CD4 T cells in the generation of robust, long lasting CD8 responses to L. monocytogenes.     

Current topics on SARS coronavirus

The serious respiratory disease, SARS (Severe Acute Respiratory Syndrome), outbreaking in winter of 2003 to 2004 remained in a sporadic patient's generating at this winter. However, there is also a possibility that wild animals as the source of infection may not be specified and that it may be much in fashion again. The paper regarding SARS and SARS-CoV is published at one per day now which has passed since fashion of SARS in one or so year. There are many papers which the researchers of other viruses enter into the research field of SARS-CoV using their own technology in addition to the researchers of coronavirus. Topics of the research on the present SARS-research field are development of vaccine, inspecting of medicine and establishment of diagnostic method. Here, the newest information is offered about these researches.     

Response of memory CD8+ T cells to severe acute respiratory syndrome (SARS) coronavirus in recovered SARS patients and healthy individuals

To date, the pathogenesis of severe acute respiratory syndrome (SARS) in humans is still not well understood. SARS coronavirus (SARS-CoV)-specific CTL responses, in particular their magnitude and duration of postinfection immunity, have not been extensively studied. In this study, we found that heat-inactivated SARS-CoV elicited recall CTL responses to newly identified spike protein-derived epitopes (SSp-1, S978, and S1202) in peripheral blood of all HLA-A*0201(+) recovered SARS patients over 1 year postinfection. Intriguingly, heat-inactivated SARS-CoV elicited recall-like CTL responses to SSp-1 but not to S978, S1202, or dominant epitopes from several other human viruses in 5 of 36 (13.8%) HLA-A*0201(+) healthy donors without any contact history with SARS-CoV. SSp-1-specific CTLs expanded from memory T cells of both recovered SARS patients, and the five exceptional healthy donors shared a differentiated effector CTL phenotype, CD45RA(+)CCR7(-)CD62L(-), and expressed CCR5 and CD44. However, compared with the high avidity of SSp-1-specific CTLs derived from memory T cells of recovered SARS patients, SSp-1-specific CTLs from the five exceptional healthy donors were of low avidity, as determined by their rapid tetramer dissociation kinetics and reduced cytotoxic reactivity, IFN-gamma secretion, and intracellular production of IFN-gamma, TNF-alpha, perforin, and granzyme A. These results indicate that SARS-CoV infection induces strong and long-lasting CTL-mediated immunity in surviving SARS patients, and that cross-reactive memory T cells to SARS-CoV may exist in the T cell repertoire of a small subset of healthy individuals and can be reactivated by SARS-CoV infection.     

Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with mycobacterial load

High Ag load in chronic viral infections has been associated with impairment of Ag-specific T cell responses; however, the relationship between Ag load in chronic Mycobacterium tuberculosis infection and functional capacity of M. tuberculosis-specific T cells in humans is not clear. We compared M. tuberculosis-specific T cell-associated cytokine production and proliferative capacity in peripheral blood from adults with progressively higher mycobacterial loads-that is, persons with latent M. tuberculosis infection (LTBI), with smear-negative pulmonary tuberculosis (TB), and smear-positive TB. Patients with smear-positive TB had decreased polyfunctional IFN-γ(+)IL-2(+)TNF-α(+) and IL-2-producing specific CD4 T cells and increased TNF-α single-positive cells, when compared with smear-negative TB and LTBI. TB patients also had increased frequencies of M. tuberculosis-specific CD8 T cells, compared with LTBI. M. tuberculosis-specific CD4 and CD8 T cell proliferative capacity was profoundly impaired in individuals with smear-positive TB, and correlated positively with ex vivo IFN-γ(+)IL-2(+)TNF-α(+) CD4 T cells, and inversely with TNF-α single-positive CD4 T cells. During 6 mo of anti-TB treatment, specific IFN-γ(+)IL-2(+)TNF-α(+) CD4 and CD8 T cells increased, whereas TNF-α and IFN-γ single-positive T cells decreased. These results suggest progressive impairment of M. tuberculosis-specific T cell responses with increasing mycobacterial load and recovery of responses during therapy. Furthermore, these data provide a link between specific cytokine-producing subsets and functional capacity of M. tuberculosis-specific T cells, and between the presence of specific CD8 T cells ex vivo and active TB disease. These data have potentially significant applications for the diagnosis of TB and for the identification of T cell correlates of TB disease progression.     

Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus

Background

Severe acute respiratory syndrome (SARS) emerged in China in 2002 and spread to other countries before brought under control. Because of a concern for reemergence or a deliberate release of the SARS coronavirus, vaccine development was initiated. Evaluations of an inactivated whole virus vaccine in ferrets and nonhuman primates and a virus-like-particle vaccine in mice induced protection against infection but challenged animals exhibited an immunopathologic-type lung disease.

Design

Four candidate vaccines for humans with or without alum adjuvant were evaluated in a mouse model of SARS, a VLP vaccine, the vaccine given to ferrets and NHP, another whole virus vaccine and an rDNA-produced S protein. Balb/c or C57BL/6 mice were vaccinated IM on day 0 and 28 and sacrificed for serum antibody measurements or challenged with live virus on day 56. On day 58, challenged mice were sacrificed and lungs obtained for virus and histopathology.

Results

All vaccines induced serum neutralizing antibody with increasing dosages and/or alum significantly increasing responses. Significant reductions of SARS-CoV two days after challenge was seen for all vaccines and prior live SARS-CoV. All mice exhibited histopathologic changes in lungs two days after challenge including all animals vaccinated (Balb/C and C57BL/6) or given live virus, influenza vaccine, or PBS suggesting infection occurred in all. Histopathology seen in animals given one of the SARS-CoV vaccines was uniformly a Th2-type immunopathology with prominent eosinophil infiltration, confirmed with special eosinophil stains. The pathologic changes seen in all control groups lacked the eosinophil prominence.

Conclusions

These SARS-CoV vaccines all induced antibody and protection against infection with SARS-CoV. However, challenge of mice given any of the vaccines led to occurrence of Th2-type immunopathology suggesting hypersensitivity to SARS-CoV components was induced. Caution in proceeding to application of a SARS-CoV vaccine in humans is indicated.     

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro

 Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8⁺ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma. Received: 4 June 1996 / Accepted: 12 November 1996     

Modular organization of SARS coronavirus nucleocapsid protein

The SARS-CoV nucleocapsid (N) protein is a major antigen in severe acute respiratory syndrome. It binds to the viral RNA genome and forms the ribonucleoprotein core. The SARS-CoV N protein has also been suggested to be involved in other important functions in the viral life cycle. Here we show that the N protein consists of two non-interacting structural domains, the N-terminal RNA-binding domain (RBD) (residues 45–181) and the C-terminal dimerization domain (residues 248–365) (DD), surrounded by flexible linkers. The C-terminal domain exists exclusively as a dimer in solution. The flexible linkers are intrinsically disordered and represent potential interaction sites with other protein and protein-RNA partners. Bioinformatics reveal that other coronavirus N proteins could share the same modular organization. This study provides information on the domain structure partition of SARS-CoV N protein and insights into the differing roles of structured and disordered regions in coronavirus nucleocapsid proteins. CK Chang and SC Sue contributed equally to this project.     

Bioinformatics analysis of SARS coronavirus genome polymorphism

Background  

We have compared 38 isolates of the SARS-CoV complete genome. The main goal was twofold: first, to analyze and compare nucleotide sequences and to identify positions of single nucleotide polymorphism (SNP), insertions and deletions, and second, to group them according to sequence similarity, eventually pointing to phylogeny of SARS-CoV isolates. The comparison is based on genome polymorphism such as insertions or deletions and the number and positions of SNPs.     

Transcutaneous anti-influenza vaccination promotes both CD4 and CD8 T cell immune responses in humans

Induction of T cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. The use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells in CD8 cell cross-priming and suggests that vaccination via the transcutaneous (TC) route may be relevant in the induction of cellular immune responses. We have previously shown that TC application of nanoparticles, on human skin explants, allows targeting of epidermal dendritic cells, possibly via hair follicles. In this study, we have investigated cellular immune responses against an influenza protein-based vaccine by TC vaccination, compared with i.m. vaccination in humans. In this study on 11 healthy volunteers, we found that a newly developed protocol based on cyanoacrylate skin surface stripping induced a significant increase in IFN-gamma-producing T cells specific for influenza vaccine by ELISPOT assays. Interestingly, TC vaccination induced both effector CD4 and CD8 T cell responses, whereas i.m. injection induced strong effector CD4 in the absence of CD8 T cells, as assessed by intracellular cytokine staining and tetramer analyses. This study proposes new perspectives for the development of vaccination strategies that trigger T cell immune responses in humans.     

Tertiary structure prediction of SARS coronavirus helicase

SARS coronavirus, SCV, has been recently responsible of a sudden and widespread infection which caused almost 800 victims. The limited amount of SCV protein structural information is partially responsible of the lack of specific drugs against the virus. Coronavirus helicases are very conserved and peculiar proteins which have been proposed as suitable targets for antiviral drugs, such as bananins, which have been recently shown to inhibit the SCV helicase in vitro. Here, the quaternary structure of SCV helicase has been predicted, which will provide a solid foundation for the rational design of other antiviral helicase inhibitors.