Anti-HBV DNA vaccination does not prevent relapse after discontinuation of analogues in the treatment of chronic hepatitis B: a randomised trial--ANRS HB02 VAC-ADN.

OBJECTIVE: The antiviral efficacy of nucleos(t)ide analogues whose main limitation is relapse after discontinuation requires long-term therapy. To overcome the risk of relapse and virological breakthrough during long-term therapy, we performed a phase I/II, open, prospective, multicentre trial using a HBV envelope-expressing DNA vaccine. DESIGN: 70 patients treated effectively with nucleos(t)ide analogues for a median of 3 years (HBV DNA <12 IU/mL for at least 12 months) were randomised into two groups: one received five intramuscular injections of vaccine (weeks 0, 8, 16, 40 and 44) and one did not receive the vaccine. Analogues were stopped after an additional 48 weeks of treatment in patients who maintained HBV DNA <12 IU/mL with no clinical progression and monthly HBV DNA for 6 months. The primary endpoint was defined as viral reactivation at week 72 (HBV DNA >120 IU/mL) or impossibility of stopping treatment at week 48. RESULTS: Reactivation occurred in 97% of each group after a median 28 days without liver failure but with an HBV DNA <2000 IU/mL in 33%; 99% of adverse reactions were mild to moderate. Immune responses were evaluated by enzyme-linked immunosorbent spot and proliferation assays: there was no difference in the percentage of patients with interferon-γ secreting cells and a specific T-cell proliferation to HBcAg but not to HBsAg after reactivation in each group. CONCLUSIONS: Although it is fairly well tolerated, the HBV DNA vaccine does not decrease the risk of relapse in HBV-treated patients or the rate of virological breakthrough, and does not restore the anti-HBV immune response despite effective viral suppression by analogues. TRIAL REGISTRATION NUMBER: NCT00536627.

Immunological and antiviral responses after therapeutic DNA immunization in chronic hepatitis B patients efficiently treated by analogues.

A substudy of a phase I/II, prospective, multicenter clinical trial was carried out to investigate the potential benefit of therapeutic vaccination on hepatitis B e antigen-negative patients with chronic hepatitis B (CHB), treated efficiently with analogues. Patients were randomized in 2 arms, one receiving a hepatitis B virus (HBV) envelope DNA vaccine, and one without vaccination. At baseline, HBV-specific interferon (IFN)-γ-producing T cells were detected in both groups after in vitro expansion of peripheral blood mononuclear cells. Vaccine-specific responses remained stable in the vaccine group, whereas in the control group the percentage of patients with HBV-specific IFN-γ-producing T cells decreased over time. The vaccine-specific cytokine-producing T cells were mostly polyfunctional CD4(+) T cells, and the proportion of triple cytokine-producer T cells was boosted after DNA injections. However, these T-cell responses did not impact on HBV reactivation after stopping analogue treatment. Importantly, before cessation of treatment serum hepatitis B surface antigen (HBsAg) titers were significantly associated with DNA or HBsAg clearance. Therapeutic vaccination in CHB patients with persistent suppression of HBV replication led to the persistence of T-cell responses, but further improvements should be searched for to control infection after treatment discontinuation.

[A most unusual foreign body in the cecum]. / Un corps étranger particulier dans le caecum.

The authors report a rare complication of total hip replacement - the intrapelvic migration of a hip prosthesis resulting in a cecal fistula.

Cellular immunity to Toxoplasma gondii in congenitally infected newborns and immunocompetent infected hosts.

The aim of this study was to determine the frequency of anergy to Toxoplasma gondii in congenitally infected newborns and immunocompetent infected individuals. Specific anergy to Toxoplasma has been reported previously, especially in cases of congenital toxoplasmosis. Whole blood from 592 immunocompetent patients with suspected toxoplasmosis was cultured in the presence of soluble Toxoplasma antigen for 7 days. Activated T lymphocytes were detected by flow cytometry. In patients over 1 year of age, the percentage of soluble Toxoplasma antigen-stimulated T cells expressing the interleukin-2 receptor CD25 was higher in infected patients than in uninfected subjects (40.0+/-18.3% vs. 1.8+/-2.0%, P<0.0001). No differences were detected between seroconverters, i.e. those with recent rises in IgM and IgG antibodies, and those with acquired or congenital toxoplasmosis. Similar results were observed when congenitally infected ( n=38) and uninfected ( n=89) children under 1 year of age were compared (17.6+/-9.2% vs. 3.0+/-4.9%, P<0.0001). The sensitivity and specificity values of CD25 detection for diagnosis of congenital toxoplasmosis in infants were 95% and 89%, respectively, at a threshold value of 7% above control culture. The results show that specific cellular immunity is detectable in virtually all Toxoplasma-infected patients, including newborns. Detection of CD25 constitutes a simple, sensitive and specific test for diagnosis of congenital toxoplasmosis.

Reduced blood CD123+ (lymphoid) and CD11c+ (myeloid) dendritic cell numbers in primary HIV-1 infection.

Successful immunologic control of HIV infection is achieved only in rare individuals. Dendritic cells (DCs) are required for specific antigen presentation to naive T lymphocytes and for antiviral, type I interferon secretion. Two major blood DC populations are found: CD11c+ (myeloid) DCs, which secrete IL-12, and CD123+ (IL-3-receptor+) DCs (lymphoid), which secrete type I interferons in response to viral stimuli. The authors have previously found a decreased proportion of blood CD11c+ DCs in chronic HIV+ patients. In this study, 26 to 57 days after infection and before treatment, CD123+ and CD11c+ DC numbers were dramatically reduced in 13 HIV+ patients compared with 13 controls (P =.0002 and P =.001, respectively). After 6 to 12 months of highly active antiretroviral therapy, DC subpopulation average numbers remained low, but CD123+ DC numbers increased again in 5 of 13 patients. A strong correlation was found between this increase and CD4 T-cell count increase (P =.0009) and plasma viral load decrease (P =.009). Reduced DC numbers may participate in the functional impairment of HIV-specific CD4+ T cells and be responsible for the low type I interferon responsiveness already known in HIV infection. The restoration of DC numbers may be predictive of immune restoration and may be a goal for immunotherapy to enhance viral control in a larger proportion of patients.

[Congenital toxoplasmosis. Transitory negative serology]. / Toxoplasmose congénitale. Négativation transitoire de la sérologie.

OBJECTIVES: Toxoplasmosis serology may become temporarily negative in children with congenital toxoplasmosis, leading to a risk of misdiagnosis and inadequate surveillance. The purpose of our work was to better understand the time course of toxoplasmosis serology which has not been studied specifically and to propose practical recommendations. PATIENTS AND METHODS: We conducted a prospective study in 217 children born with congenital toxoplasmosis between January 1988 and December 1997. Clinical, ophthalmological and serology data were collected every three months during their first year of life then every six months until three years of age and every year thereafter for all patients. Negative serology was defined as the absence of IgG at indirect immunofluorescence and ELISA (enzyme linked immunosorbent assay) and by the absence of IgM at ISAGA (immunosorbent agglutination assay). RESULTS: During the mean follow-up of 66 +/- 33 months (range 12-126 months), 33 children (15%) presented a period where the toxoplasmosis serology (ELISA and indirect immunofluorescence) was negative for a transient period reaching a mean 5 months. The dye test was performed in 25 of these children and was negative in 6 (24%). Among the negative conversions observed at routine testing, 73% occurred in children taking pyrimethamine/sulfadoxin therapy and the others occurred a mean 11.7 months after interruption of treatment. There was a positive association between maternal treatment and transient seronegativity in the cases where the maternal contamination had occurred during the first 2 trimesters of pregnancy. The serology became positive again in 30 of the 33 children (91%) and in 22 children there was a rebound. At last follow-up, the 3 other children still had negative serology (mean duration 35 months, range 3-62 months). CONCLUSION: Transient negative toxoplasmosis serology is a frequent phenomenon in children with congenital toxoplasmosis. Although the underlying pathophysiological mechanism remains unknown, it is crucial to avoid questioning the initial diagnosis of congenital toxoplasmosis and to continue regular routine monitoring.

A switch towards Th2 during serological rebound in children with congenital toxoplasmosis.

Serological rebounds occur frequently in patients with congenital toxoplasmosis, but remain poorly understood. A link between Th1 and Th2 cytokines and the pathophysiology of infectious diseases has been reported. Production of interferon-gamma (IFN-gamma) and IL-4 in supernatants of whole blood after in vitro specific Toxoplasma gondii stimulation and serum-specific IgE levels were studied in 31 congenitally infected children. IFN-gamma was produced at higher levels by lymphocytes from children with stable congenital toxoplasmosis (n = 18) than from children showing serological rebound (n = 13) (P < 0.04). Conversely, supernatants from children with serological rebound showed higher levels of IL-4 than those from children with stable congenital toxoplasmosis (P < 0.03). The polarized Th2 response was confirmed by a greater (IL-4:IFN-gamma) x 100 ratio (P < 0.0001) and production of T. gondii-specific IgE in six out of 13 children showing serological rebound. These results suggest a role of Th2 cytokines in destabilization of congenital toxoplasmosis and perhaps in local reactivation of the parasite.

Flow cytometric quantification of Toxoplasma gondii cellular infection and replication.

The invasion and replication of Toxoplasma gondii are usually analyzed through either optical microscopy or incorporation of tritiated uracil. A new method has been developed using flow cytometric analysis to examine the entry and replication of T. gondii RH strain in Saimiri brain endothelial cells. After cell fixation and permeabilization using saponin, intracellular T. gondii were labeled with a monoclonal antibody against T. gondii SAG-1 (P30; the major cell-surface antigen) followed by fluorescein-conjugated rabbit anti-mouse IgG. The percentage of infected cells obtained using flow cytometry correlated directly with that obtained by UV light microscopy (r = 0.97). The mean fluorescence intensity of infected cells reflects intracellular P30 and assesses intracellular replication. The distribution of fluorescence per infected cell, considered with the percentage of infected cells, also allows a qualitative analysis of replication. Such a method is rapid, easy, and does not require specialized equipment for radioactive labeling.

A rapid flow cytometric method to explore cellular immunity against Toxoplasma gondii in humans.

To assess cell-mediated immunity to Toxoplasma gondii, we evaluated the expression of the activation antigens CD69, CD71, and CD25 on T lymphocytes by flow cytometry after specific in vitro stimulation of whole blood from 127 T. gondii-positive and 63 T. gondii-negative patients. T lymphocytes from many seropositive individuals did not express CD69 at 24 h after T. gondii antigen stimulation, but CD71 and CD25 were easily detectable on T cells from seropositive individuals 7 days after specific activation. CD25 was mainly expressed by stimulated CD4(+) T cells, and its detection on total T cells was both a sensitive (98%) and a specific (97%) indicator of prior T. gondii infection. These results make flow cytometric detection of CD25 an excellent candidate for screening cell-mediated immunity to T. gondii in vitro and an interesting tool for the diagnosis of congenital infection.

Circulating Toxoplasma gondii-specific antibody-secreting cells in patients with congenital toxoplasmosis.

Patients with congenital toxoplasmosis occasionally show rises in serum antibodies to Toxoplasma gondii (serological rebound), but the underlying cause remains unclear. The acute or chronic presence of available antigen often causes the appearance, in the peripheral blood, of cells actively secreting specific antibody. We have evaluated the capacity of circulating blood cells from 91 children born to T. gondii-infected mothers to actively synthesize anti-T. gondii antibodies according to their serological status. Supernatants from 7-day cultures of peripheral blood mononuclear cells were evaluated for antibody by cytofluorimetry. Only 1 of 49 subjects with low and stable serum antibody titers produced specific antibodies on cultures, while 9 of 22 subjects with recent rebound were positive. One of the positive children alone showed clinical signs of parasite activity. These observations suggest that rebound may be associated with production of available parasite antigens, possibly associated with reactivation. Differentiation from other causes, such as polyclonal B cell stimulation, would improve our ability to detect clinically significant reactivation and to prevent complications.