Enhancement of human papillomavirus (HPV) type 16 E6 and E7-specific T-cell immunity in healthy volunteers through vaccination with TA-CIN, an HPV16 L2E7E6 fusion protein vaccine.

TA-CIN is a vaccine that comprises the human papillomavirus (HPV) type 16 L2, E6 and E7 as a single fusion protein. In a mouse model, TA-CIN effectively prevented outgrowth of HPV16-positive tumour cells. To assess the safety and immunogenicity of TA-CIN, a dose escalating (26, 128, 533 micro g), double blind and placebo-controlled phase I study was conducted in 40 healthy volunteers. TA-CIN was administered without adjuvant by intramuscular injection on weeks 0, 4 and 8. No serious adverse events of the vaccination were reported during the study. Both IgG antibodies and proliferative responses against TA-CIN were elicited at all three doses. More importantly, T-cell immunity against the HPV16 E6 and E7 oncoproteins was detected by IFN gamma ELISPOT in 8/11 evaluable subjects vaccinated with the 533 micro g dose.

Novel Salmonella enterica serovar Typhimurium protein that is indispensable for virulence and intracellular replication.

Upon contact with host cells, the intracellular pathogen Salmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribed Salmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonella chromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 10(4) to 10(7) bacteria in C3H/HeN and 10(1) to 10(4) bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.

Pre-clinical safety and efficacy of TA-CIN, a recombinant HPV16 L2E6E7 fusion protein vaccine, in homologous and heterologous prime-boost regimens.

Human papillomavirus (HPV) E6 and E7 oncoproteins are attractive targets for T-cell-based immunotherapy of cervical intraepithelial neoplasia (CIN) and cancer. A newly designed vaccine, comprising the HPV16 L2, E6 and E7 as a single fusion protein (TA-CIN), was shown to elicit HPV16-specific CTL, T-helper cells and antibodies in a pre-clinical mouse model. These immune responses effectively prevented outgrowth of HPV16-positive tumour cells in a prophylactic setting as well as in a minimal residual disease setting. CTL immunity was optimally induced when TA-CIN was employed in heterologous prime-boost regimens in combination with TA-HPV, a clinical grade vaccinia-based vaccine. These data provide a scientific basis for the use of TA-CIN, alone or in combination with TA-HPV in future human trials.

Natural T-helper immunity against human papillomavirus type 16 (HPV16) E7-derived peptide epitopes in patients with HPV16-positive cervical lesions: identification of 3 human leukocyte antigen class II-restricted epitopes.

Tumor-specific T-helper (Th) immunity was found to play a pivotal role in the natural and vaccine-induced immune defense against tumors. Since the majority of cervical cancers express human papillomavirus type 16 (HPV16) E7 oncoprotein, it is important to investigate the Th response against this target antigen in detail. By means of PBMC cultures from HLA-typed healthy donors, we identified the central part of HPV16 E7 (E7(41-72)) as the major immunogenic region within this antigen. Furthermore, we mapped 3 distinct Th epitopes within this region (DR15/E7(50-62), DR3/E7(43-77), DQ2/E7(35-50)). In a parallel approach, employing IFN-gamma ELISPOT analysis, we detected Th immunity against HPV16 E7 in subjects with HPV16+ lesions. Several of these responses matched with the 3 Th epitopes defined in our study. A number of other HPV16+ subjects did not display any E7-specific type 1 cytokine-producing T-cell immunity, indicating failure of the immune response. Our combined data argue for more extensive as well as longitudinal analysis of HPV16-specific T-cell immunity using the ELISPOT assay described, as well as for HPV-specific vaccination of individuals with HPV+ lesions.

Molecular mimicry of human cytochrome P450 by hepatitis C virus at the level of cytotoxic T cell recognition.

Hepatitis C virus (HCV) is thought to be involved in the pathogenesis of autoimmune hepatitis (AIH) type 2, which is defined by the presence of type I antiliver kidney microsome autoantibodies directed mainly against cytochrome P450 (CYP)2D6 and by autoreactive liver infiltrating T cells. Virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) that recognize infected cells and contribute to viral clearance and tissue injury during HCV infection could be involved in the induction of AIH. To explore whether the antiviral cellular immunity may turn against self-antigens, we characterized the primary CTL response against an HLA-A*0201-restricted HCV-derived epitope, i.e., HCV core 178-187, which shows sequence homology with human CYP2A6 and CYP2A7 8-17. To determine the relevance of these homologies for the pathogenesis of HCV-associated AIH, we used synthetic peptides to induce primary CTL responses in peripheral blood mononuclear cells of healthy blood donors and patients with chronic HCV infection. We found that the naive CTL repertoire of both groups contains cross-reactive CTLs inducible by the HCV peptide recognizing both CYP2A6 and CYP2A7 peptides as well as endogenously processed CYP2A6 protein. Importantly, we failed to induce CTLs with the CYP-derived peptides that showed a lower capacity to form stable complexes with the HLA-A2 molecule. These findings demonstrate the potential of HCV to induce autoreactive CD8(+) CTLs by molecular mimicry, possibly contributing to virus-associated autoimmunity.

Identification of a conserved universal Th epitope in HIV-1 reverse transcriptase that is processed and presented to HIV-specific CD4+ T cells by at least four unrelated HLA-DR molecules.

CD4+ Th cells play an important role in the induction and maintenance of specific T cell immunity. Indications for a protective role of CD4+ T cells against HIV-1 infection were found in subjects who were able to control HIV-1 viremia as well as in highly HIV-1-exposed, yet seronegative, individuals. This study describes the identification of an HIV-1-specific Th epitope that exhibits high affinity binding as well as high immunogenicity in the context of at least four different HLA-DR molecules that together cover 50-60% of the Caucasian, Oriental, and Negroid populations. This HIV-1 reverse transcriptase-derived peptide (RT171-190) is highly conserved among different HIV-1 isolates. Importantly, stimulation of PBL cultures from HIV-1 seronegative donors with this peptide resulted in Thl-type lymphocytes capable of efficient recognition of HIV-1-pulsed APCs. Taken together, these data indicate that peptide RT171-190 constitutes an attractive component of vaccines aiming at induction or enhancement of HIV-1-specific T cell immunity.

Anti-inflammatory cytokine profile and mortality in febrile patients.

BACKGROUND: An anti-inflammatory cytokine profile on whole-blood stimulation in vitro is associated with fatal outcome of meningococcal disease. We investigated whether an anti-inflammatory cytokine profile in the circulation is associated with adverse outcome in other infectious diseases. METHODS: We enrolled 464 consecutive patients (272 men, 192 women) who presented to hospital with fever (> or = 38.2 degrees C). On admission we measured plasma interleukin 10 (IL-10) and tumour necrosis factor alpha (TNF alpha), and collected clinical and microbiological data on the febrile illness, then followed up all patients for clinical outcome. FINDINGS: In at least 399 of the 464 patients fever was caused by infection. 33 patients died after a median hospital stay of 11 days (interquartile range 3-20). Concentrations of IL-10 were significantly higher in non-survivors (median 169 pg/mL [IQR 83-530]) than in survivors (median 88 pg/mL [42-235], p=0.042). When dichotomised around the median, the mortality risk was two times higher in patients who had high concentrations of IL-10 than in those with low concentrations (relative risk 2.39 [95% CI 1.07-5.33]), in patients with low and high concentrations of TNF alpha. In the 406 patients without haemodynamic deterioration in the first 24 h, IL-10 was higher and TNF alpha lower in patients who died than in those who survived. The ratio of IL-10 to TNF alpha was higher in non-survivors (median 6.9 [3.0-21.0]) than in survivors (median 3.9 [2.0-7.0], p=0.040). This ratio was highest in patients who died without underlying disease (median 21.5 [5.0-25.0]). Age, sex, and duration of fever before admission did not explain the differences in IL-10 and TNF alpha. INTERPRETATION: An anti-inflammatory cytokine profile of a high ratio of IL-10 to TNF alpha is associated with fatal outcome in febrile patients with community-acquired infection. Our findings caution against a widespread use of proinflammatory cytokine inhibition in patients with sepsis.

Antibiotic-induced lipopolysaccharide (LPS) release from Salmonella typhi: delay between killing by ceftazidime and imipenem and release of LPS.

It has been suggested that the antibiotic-induced release of lipopolysaccharide (LPS) is an important cause of the development of septic shock in patients treated for severe infections caused by gram-negative bacteria. Beta-lactam antibiotics change the integrity of the bacterial cell envelope by binding to penicillin-binding proteins (PBP) in the membrane and thus may affect the amount of LPS that is released and the kinetics of that release. In this respect, ceftazidime at intermediate concentrations binds with a high affinity to PBP 3 and PBP 1a and thus can induce filament formation in addition to killing, whereas imipenem preferentially binds to PBP 2 and PBP 1b, leading to spheroplast formation and rapid cell lysis. We investigated the effects of these antibiotics on the killing and the release of the radioactively labelled LPS of Salmonella typhi Ty 21A. A mathematical model was developed to calculate the delay between bacterial killing and LPS release, designated the lag time. At antibiotic concentrations inducing equal killing, the amount of LPS released was the same for both antibiotics. Only after 6 h of incubation at antibiotic concentrations above 0.5 microg/ml, the amount of 3H-LPS released was slightly higher (approximately 1.2-fold) in incubations with ceftazidime than in those with imipenem, and the maximum releases of the total label were 33.2% +/- 0.89% and 27.1% +/- 0.45%, respectively. Despite the clear concentration-dependent effect on the bacterial killing and subsequent LPS release, the lag time was independent of the antibiotic concentration. For ceftazidime as well as imipenem the lag time amounted to approximately 60 min. In conclusion, our findings imply that the mechanism of antibiotic-induced LPS release is independent of the PBP affinities for these beta-lactam antibiotics. Furthermore, once the organism is killed by either imipenem or ceftazidime, the rate of LPS release from S. typhi does not differ according to the antibiotic with which the organism is killed, and there is little difference in the relative amount of LPS released.

Relation between pro- and anti-inflammatory cytokines and the production of nitric oxide (NO) in severe sepsis.

The aim of the present study was to investigate the relationship between the levels of pro-inflammatory [interleukin 6 (IL-6), IL-8, tumour necrosis factor alpha (TNF-alpha)], anti-inflammatory cytokines [IL-10, soluble TNF receptor type I (TNFsrI), TNFsrII], and the production of nitric oxide (NO) during a 1-week period in 23 patients with severe sepsis. The highest levels of pro-inflammatory cytokines and nitrate, the stable metabolite of NO, were found during the first day after inclusion and gradually declined thereafter. Detectable levels of IL-10, TNFsrI and TNFsrII were present in all patients at study entry but did not significantly change during the study period [analysis of variance (MANOVA); P > 0.05]. Serum nitrate levels correlated significantly with both pro-inflammatory cytokines (IL-6, IL-8, TNF-alpha) as well as anti-inflammatory cytokines (IL-10, TNFsrI, TNFsrII). Serum nitrate levels over time were higher in patients with positive blood cultures (n = 4) (MANOVA; P < 0.005), as compared to patients without proven bacteraemia. These data support the concept of an acute phase of sepsis that is characterized by an excess of pro-inflammatory cytokines, while anti-inflammatory cytokines are predominantly present during the secondary phase. The present findings indicate that pro-inflammatory cytokines are related to the induction of excessive NO production during the first phase of sepsis and that reduction of NO production occurs during the secondary phase. This may suggest that anti-inflammatory cytokines are able to diminish the production of NO in patients with severe sepsis.

Nitric oxide (NO) production correlates with renal insufficiency and multiple organ dysfunction syndrome in severe sepsis.

OBJECTIVE: To investigate whether the production of nitric oxide (NO) relates to the development of renal insufficiency and multiple organ dysfunction syndrome (MODS) in patients with severe sepsis. DESIGN: Prospective study in 23 patients with severe sepsis. SETTING: Medical and surgical intensive care units (ICU) of three hospitals. MEASUREMENTS AND RESULTS: Serum nitrate levels, as an indirect parameter of the production of NO in vivo, and scores for renal insufficiency and MODS were determined in patients with severe sepsis during a 1-week period after admission to the ICU. The highest serum nitrate levels were found at 4 h (mean 52 +/- 16 mumol/l) after entry into the study and the levels gradually declined thereafter. Patients with renal insufficiency had considerably higher serum nitrate levels during the study period that patients who did not develop renal sufficiency (MANOVA, p < 0.05). Serum nitrate levels correlated with scores for renal insufficiency (r = 0.60, p < 0.001), and far exceeded the levels that can be explained solely by reduced renal clearance of nitrate. Further analysis showed that serum nitrate levels significantly and positively correlated with scores for MODS (r = 0.44, p < 0.001). CONCLUSION: Our results indicate that the production of NO correlates with renal insufficiency and MODS in patients with severe sepsis and that this reactive nitrogen intermediate could be involved in the pathogenesis of organ failure in these critically ill patients.

S. typhi vaccine strain Ty21a can cause a generalized infection in whole body-irradiated but not in hydrocortisone-treated mice.

Various mutations including galE- in the S.typhi vaccine strain Ty21a are thought to prevent proliferation of these micro-organisms in the host, and elimination of Ty21a would occur independent of the immune system of the host. To investigate this issue, we determined whether Ty21a can proliferate in immunosuppressed mice, and assessed the role of phagocytes in the eradication of Ty21a from tissues. Mice were rendered lymphocytopenic and monocytopenic by hydrocortisone s.c., or were made leucocytopenic by whole body irradiation. Bacteria were injected into a tail vene to evaluate eradication from the blood, liver and spleen, and into thigh muscle, i.e. a tissue that lacks resident macrophages. Ty21a were grown overnight in glucose [glu], or galactose and glucose [gal.glu]; only the Ty21a [gal.glu] expressed somatic O-antigens. After i.v. injection of 10(4) to 10(6) micro-organisms, Ty21a were rapidly eliminated from the liver and spleen of normal and immunosuppressed mice, i.e. within 1 day a 95% reduction of bacterial counts was observed. After i.m. injection of 10(4) to 10(6) bacteria, the number of viable Ty21a decreased in normal and hydrocortisone-treated mice, but in irradiated mice the micro-organisms proliferated and caused generalized infection. In all cases, Ty21a [glu] was eliminated more rapidly than Ty21a [gal.glu], confirming reports that killing of bacteria that lack O-antigens is more rapid than that of smooth bacteria of the same species. These results indicate that elimination of the vaccine strain against typhoid fever, Ty21a, from host tissues is not due to an intrinsic property of the micro-organisms that prevents proliferation but instead depends on the action of resident macrophages and exudate monocytes and granulocytes.

Increased production of nitric oxide in patients infected with the European variant of hantavirus.

Serum nitrate levels, a measure of nitric oxide (NO) production in vivo, were very high (95 +/- 14 microM) in 13 patients infected with Puumala virus, the European variant of Hantavirus (HTV), as compared to those in healthy subjects (33 +/- 3 microM). Serum nitrate levels showed a high and significant correlation with scores on the Acute Physiological And Chronic Health Evaluation (APACHE II) scale and with serum creatinine, and an inverse correlation with platelet counts. Serial serum measurements of nitrate in 2 severe cases showed very high levels at the onset of arterial hypotension and acute renal failure. We conclude that excessive amounts of NO are produced in patients infected with Puumala virus and that this reactive nitrogen intermediate could play an important role in the pathogenesis of the disease.