ABSTRACT To evaluate whether the rectal route of immunization may be used to provide appropriate protection against enteric pathogens such as rotaviruses (RV), we studied the antibody response and the protection induced by rectal immunization of mice with RV virus-like particles (VLP). For this purpose, 6-week-old BALBc mice were rectally immunized twice with RV 8-2/6/7-VLP derived from the bovine RV RF81 strain either alone or combined with various adjuvants including four toxins [cholera toxin (CT) and three attenuated Escherichia coli-derived heat-labile toxins (LTs), LT(R192G), LT(R72), and LT(K63)] and two Toll-like receptor-targeting adjuvants (CpG and resiquimod). Six weeks after the second immunization, mice were challenged with murine RV strain ECw. RV VLP administered alone were not immunogenic and did not protect mice against RV challenge. By contrast, RV VLP combined with any of the toxin adjuvants were immunogenic (mice developed significant titers of anti-RV immunoglobulin A [IgA] in both serum and feces and of anti-RV IgG in serum) and either efficiently induced complete protection of the mice (no detectable fecal virus shedding) or, for LT(K63), reduced the amount of fecal virus shedding after RV challenge. When combined with RV VLP, CpG and resiquimod failed to achieve protection, although CpG efficiently induced an antibody response to RV. These results support the consideration of the rectal route for the development of new immunization strategies against RV infection. Rectal delivery of a VLP-based vaccine might allow the use of adjuvants less toxic than, but as efficient as, CT.

BACKGROUND: WBC depletion by filtration may prevent the transmission of HTLV‐I, which requires cell‐to‐cell contact. The removal of HTLV‐I‐infected cells in routinely filtered blood cell components was measured.STUDY DESIGN AND METHODS: The study was conducted in Martinique where systematic screening for HTLV‐I and ‐II and universal leukoreduction are mandatory. HTLV‐I was quantified by use of real‐time PCR in 8 RBC units and 4 PLT concentrates before and after filtration. HTLV‐I proviral load in PBMNCs was determined in five of the eight HTLV‐I‐infected blood donors.RESULTS: The amount of MNC‐associated HTLV‐I DNA in RBC units before filtration was 21 × 106± 29 × 106 copies (mean ± SD). HTLV‐I was detected in 4 of 8 RBC units after filtration, with a number of copies in the MNC fraction ranging from 20 to 140, following a 4.9 to 5.8 log reduction. Flow cytometry analysis performed in 2 of the filtered RBC units containing detectable HTLV‐I showed suboptimal and out‐of‐range leukoreduction (0.56 × 106 and 1.22 × 106 residual WBCs). HTLV was not detected in filtered RBCs from the blood donor with the highest percentage of HTLV‐I‐infected PBMCs (9%).CONCLUSION: This study confirms that HTLV‐I‐infected cells can be detected in filtered blood cell components and shows that optimal leukoreduction is critical for HTLV‐I removal.

A quantitative real-time PCR assay was developed to measure the proviral load of human T-lymphotropic virus type I (HTLV-I) in peripheral blood mononuclear cells (PBMCs). The HTLV-I copy number was referred to the actual amount of cellular DNA by means of the quantitation of the albumin gene. Ten copies of HTLV-I DNA could be detected with 100% sensitivity, and the assay had a wide range of at least 5 log(10). Intra- and inter-assay reproducibility was evaluated using independent extractions of PBMCs from an HTLV-I-infected patient (coefficients of variation, 24 and 7% respectively). The performance of this TaqMan PCR assay, coupled with its high throughput, thus allows reliable routine follow-up of HTLV-I proviral load in infected patients. Preliminary results using clinical samples indicate a higher proviral load in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis than in asymptomatic carriers, and also suggest the usefulness of this quantitative measurement to assess the etiological link between HTLV-I and adult T-cell leukaemia/lymphoma-like syndromes.

Several reports suggest that HTLV-I/HIV coinfection may be associated with an increased risk of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). In HTLV-I-monoinfected patients, the occurrence of HAM/TSP is associated with high peripheral blood HTLV-I proviral load. Using a real-time quantitative PCR assay, we assessed the proviral DNA load in peripheral blood mononuclear cells (PBMCs) from 15 asymptomatic HTLV-I-monoinfected patients, 15 HTLV-I-monoinfected patients with HAM/TSP, and 25 HTLV-I/HIV-1 coinfected patients, including 4 with HAM/TSP. We also measured HIV-1 proviral DNA load in PBMCs from the coinfected patients. The median HTLV-I proviral loads were 6,800 and 4,100 copies per 106 PBMCs in the asymptomatic monoinfected and coinfected groups, and 58,800 and 43,300 copies per 106 PBMCs in the monoinfected and coinfected patients with HAM/TSP, respectively. The difference between HTLV-I proviral loads in HAM/TSP and asymptomatic monoinfected patients was statistically significant (p < 0.0001), but there was no difference between the HTLV-I-monoinfected and HTLV-I/HIV-1-coinfected groups. There was no correlation between HTLV-I and HIV-1 proviral load. HTLV-I proviral load did not correlate with the CD4+ T lymphocyte count. Among patients with no HTLV-I disease, the median copy number of HTLV-I per 106 circulating CD4+ T cells was 114,000 in the coinfected group and 16,700 in the monoinfected group, but the difference was not significant (p = 0.089). These data do not confirm the hypothesis in which HIV-1 coinfection would increase HTLV-I proviral burden in the PBMCs. However, depletion of the CD4+ T cell subset, the main target of HTLV-I, could be counterbalanced by an up-regulation of HTLV-I replication or by greater resistance of HTLV-I-infected cells to HIV-1-induced destruction.

ABSTRACT Proviral human immunodeficiency virus type 1 (HIV-1) DNA could be a useful marker for exploring viral reservoirs and monitoring antiretroviral treatment, particularly when HIV-1 RNA is undetectable in plasma. A new technique was developed to quantify proviral HIV-1 using a TaqMan real-time PCR assay. One copy of proviral HIV-1 DNA could be detected with 100% sensitivity for five copies and the assay had a range of 6 log 10 . Reproducibility was evaluated in intra- and interassays using independent extractions of the 8E5 cell line harboring the HIV-1 proviral genome (coefficients of variation [CV], 13 and 27%, respectively) and peripheral blood mononuclear cells (PBMC) from a patient with a mean proviral load of 26 copies per 10 6 PBMC (CV, 46 and 56%, respectively). The median PBMC proviral load of 21 patients, measured in a cross-sectional study, was determined to be 215 copies per 10 6 PBMC (range, <10 to 8,381). In a longitudinal study, the proviral load of 15 out of 16 patients with primary infection fell significantly during 1 year of antiretroviral therapy ( P = 0.004). In the remaining patient, proviral HIV-1 DNA was detectable but not quantifiable due to a point mutation at the 5′ end of the TaqMan probe. No correlation was observed between proviral load and levels of CD4 + cells or HIV-1 RNA in plasma. TaqMan PCR is sensitive and adaptable to a large series of samples. The full interest of monitoring proviral HIV-1 DNA can now be ascertained by its application to the routine monitoring of patients.

There is a general consensus on the need to start antiretroviral therapy rapidly after the diagnosis of HIV infection, although the optimal drug combinations and duration of treatment are unknown [1]. We treated nine patients with HIV-1 subtype B primary infection with a triple nucleoside analogue combination (zidovudine, didanosine and lamivudine) for 12 months, and examined the effect of treatment discontinuation. Treatment was resumed when the plasma viral load reached 20 000 copies/ml. A marked decrease in plasma HIV RNA was observed rapidly after treatment initiation. The HIV-RNA level was below 500 copies/ml at month 3 in all the patients, and below 50 copies/ml at month 6 in all but one of the patients (patient no. 5, 76 copies/ml) (Fig. 1). The plasma HIV-RNA level remained below 500 copies/ml in all patients at month 12, and the mean reduction in HIV-RNA levels at month 12 compared with the baseline was 2.5 ± 0.6 log10 copies/ml (1.76–3.7;P = 0.008, Wilcoxon's matched-pairs signed rank test). Cellular HIV viraemia tended to fall below the detection limit later than plasma HIV RNA. Samples were available for proviral DNA quantification (using the TaqMan technique) in seven patients. A significant decrease was observed on treatment, from a mean of 1810 ± 3276 copies/106 peripheral blood mononuclear cells (PBMC) on day zero to 118 ± 133 copies/106 PBMC at month 12 (P = 0.018, Wilcoxon's matched-pairs signed rank test).Fig. 1.: Serial measurements of circulating HIV. The one year period of triple nucleoside analogue therapy is indicated by the grey line at the top of each figure, whereas the blank line indicates treatment discontinuation. Open squares indicate plasma HIV-RNA levels (lower quantification limit 500 copies/ml), and +/− indicates detectability in a more sensitive test (detection limit 50 copies/ml). Open circles indicate cellular viraemia (detection limit 0.1 IU/106 peripheral blood mononuclear cells; PBMC). Open triangles indicate proviral HIV-DNA levels (detection limit 10 copies/106 PBMC). Results are given up to month 30 in the five patients who remained off therapy, and until treatment re-introduction in the remaining four patients. Day zero samples for HIV DNA proviral measurement were not available in two cases (patient nos. 1 and 2).Antiretroviral therapy was discontinued in all patients at month 12. HIV-RNA levels again became detectable (> 50 copies/ml) in all patients one month after treatment discontinuation, with a maximal value of 29 280 copies/ml. However, plasma HIV-RNA levels remained low (below 500 copies/ml) in three patients for 6 months (patient nos. 1 and 2) and 18 months (patient no. 3). Treatment was resumed in four patients at month 13 (patient no. 6; viral load: 29 280 copies/ml), month 14 (patient no. 7; viral load: 27 220 copies/ml) and month 15 (viral load: 88 647 copies/ml in patient no. 9 and 99 780 copies/ml in patient no. 8). In the five patients who were not retreated, HIV-RNA levels stabilized at values of less than 500 to 12 395 copies/ml until the last evaluation (month 30), 18 months after treatment discontinuation. Proviral DNA was undetectable at month 30 in one of these five patients (no. 3), detectable but below the threshold of quantification in two patients (nos. 2 and 4), and 90 and 361 copies/106 PBMC in the remaining two patients (nos. 1 and 5). Three of these patients had a fully reactive Western blot, one (two patients) and 3 months (one patient) after treatment discontinuation. The HIV reverse transcriptase gene was sequenced on day zero and at the first viral rebound after treatment discontinuation. In day zero samples, three patients had zidovidune resistance-associated mutations (M41L: no. 4; K70R: no. 9; K70R plus T215Y: no. 6). These mutations persisted on viral rebound at month 13. An acquired lamivudine resistance-associated mutation (M184V) was detected on viral rebound at month 13 in three patients (nos. 5, 6 and 9). Two of these patients (nos. 6 and 9) had zidovudine resistance-associated mutations at day zero. The plasma viral load remained below 50 copies/ml during therapy in one patient (no. 6), and was between 50 and 500 copies/ml in the other two patients (nos. 5 and 9). The M184V mutations were no longer detectable at month 15. The V3V4 genomic region exhibited no sequence variations in the six patients who were tested between day zero and month 12. There was a significant gradual increase in the CD4 cell count during treatment, with a mean gain at month 12 of 206 ± 184/mm3 (range −50 to +520/mm3;P = 0.021), whereas the CD8 cell count fell initially (month 1) and then remained stable. There was a trend towards a slight reduction in the CD4 and CD8 cell counts when the patients were off therapy (−114 ± 96 and −54 ± 191/mm3, respectively). At month 24, T cell proliferative responses to tuberculin were highly positive in eight out of nine patients, whereas responses to cytomegalovirus and tetanus toxoid were only positive in one patient each. No reactivity to HIV-1 p24 antigen was observed, even in patients with a stable viral load. Patients were re-tested at month 36, and no improvement in T cell functions was observed. The resumption of antiretroviral therapy did not modify immune responses compared with patients who remained off therapy. The rapid and marked reduction in HIV-RNA load observed in our study is in keeping with results obtained with other antiretroviral combinations [2,3]. The reduction in HIV cell viraemia followed a pattern similar to that in plasma HIV RNA. The M184V mutation arose in three patients within the first year of therapy. Interestingly, two of them had small sporadic rebounds of plasma HIV-RNA levels during therapy (between 50 and 500 copies/ml). The lack of variations in the predominant V3V4 sequence from day zero to month 12 in the six assessable patients must be interpreted with care. Cloning was performed at day zero and month 12 in only one patient (no. 5): no sequence variations were found in the eight and 11 clones obtained at day zero and month 12, respectively. Despite the lack of V3V4 variability, the M184V mutation in the reverse transcriptase gene occurred in this patient and two others, pointing to persistent replication during treatment. Although all the patients had active viral replication after treatment discontinuation, the level of plasma HIV RNA was initially low and increased slowly in five out of nine patients (≤ 12 395 copies/ml ) until month 30. In addition, plasma HIV-RNA levels remained between 50 and 500 copies/ml in one patient during 18 months off therapy. This contrasts with the situation observed in patients with advanced HIV infection, in whom the viral rebound after the withdrawal of a protease inhibitor-containing combination reached pretreatment levels or higher after only one week [4,5]. Lymphocyte proliferation results were disappointing in our study, as no reactivity to HIV-1 p24 antigen developed during the period off therapy. In conclusion, this uncontrolled study shows the effectiveness and good tolerability of first-line triple nucleoside analogue therapy for patients with primary HIV infection. Treatment interruption after 12 months was associated with low-level HIV replication in five out of nine patients. Given the inability of current treatments to eradicate HIV, treatment discontinuation after an initial one year course may be an alternative to continuous life-long therapy [6,7]. Pierre-Marie Girarda Véronique Schneiderb Axelle Dehéeb Philippe Mariota Christine Jacometa Nathalie Delphinb Florence Damondc Guislaine Carcelaine Brigitte Autrane Adrien Gérard Saimotd Jean-Claude Nicolasb Willy Rozenbauma

Epstein‐Barr virus (EBV) reactivation is more likely to occur in immunocompromised patients with subsequent higher susceptibility to EBV‐associated lymphoproliferations. In contrast to transplant recipients, limited data are available concerning the EBV load in HIV‐infected patients, with or without AIDS‐related non‐Hodgkin's lymphomas. We developed a TaqMan real‐time PCR assay, allowing both the EBV genome and a cellular gene to be quantified in order to obtain a reliable normalized measurement of the EBV load in peripheral blood mononuclear cells (PBMCs). With a wide 6‐log10 quantification range and inter‐assay variations of less than 24%, this quantitative PCR was sufficiently accurate and reproducible for routine follow‐up. The EBV load was determined in PBMCs from 113 HIV‐infected patients, 11 patients with primary HIV infection and 24 HIV‐seronegative healthy controls. The rates of EBV detection were similar in the three groups. However, EBV loads were higher in the HIV‐infected group (P < 0.00001) except for the patients with primary HIV infection. Unexpectedly, EBV loads were not correlated with the clinical stages of HIV infection or HIV replication, and did not depend on the degree of immunodepression, as judged by CD4+ counts. This study contributes towards the definition of the baseline EBV load during HIV infection and stresses the broad inter‐individual variability of the EBV load in HIV‐infected patients. Real‐time PCR provides a useful tool that can be used in further longitudinal studies to assess the relevance of the EBV load to identify HIV‐infected patients with a high risk of EBV‐associated lymphoproliferations. J. Med. Virol. 65:543–552, 2001. © 2001 Wiley‐Liss, Inc.

A quantitative real-time PCR assay was developed to measure human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes (PBLs). The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin). The results of the real-time PCR assay correlated with those of the HCMV pp65-antigenemia assay (P < 0.0001).

Post-transplant lymphoproliferative disorder (PTLD) after haemopoietic stem cell transplantation is a serious complication that occurs in 8-22% of patients with high-risk factors. We retrospectively investigated tolerance and efficacy of humanized anti-CD20 monoclonal antibody (rituximab) as first-line treatment in 12 children with B-cell PTLD. At diagnosis, eight patients had tumoral involvement. The other four patients had fever, associated with raised Epstein-Barr virus (EBV) viral load and monoclonal gammopathy. Rituximab was given at the dose of 375 mg/ m(2) once a week by intravenous infusion (1-9 infusions). Only 1/48 infusions was associated with a grade 2 clinical adverse event. Eight out of 12 (66%) patients responded to the treatment and were in complete remission. All patients without tumoral involvement responded to the treatment. A rapid decrease in fever within 1 week was observed in all responders. Non-responders did not show any clinical response during the first week. Tumoral involvement and immunodepression seemed to be more marked in nonresponders. Rituximab was an effective and well-tolerated treatment of B-cell PTLD. Early treatment before tumoral involvement seemed to be the most effective approach. Lack of rapid response should lead to intensification of PTLD treatment. Pre-emptive treatment should be considered and evaluated in further longitudinal multicentre studies.

We have developed a quantitative RTPCR method that can be used to determine the amount of enterovirus RNA in urban sludge samples. This method combines Taq-Man® technology with the ABI PrismTM 7700 real-time sequence detection system. We optimized a one-step RT-PCR that uses a dual-labeled fluorogenic probe to quantify the 5′ noncoding region of enteroviruses. For accurate quantification of the number of copies, a Mahoney type 1 poliovirus RNA standard was designed and produced using genetic engineering. This fragment, quantified using the Ribogreen® method, was used in serial dilutions as an external standard. The method had a 7-log dynamic range (5 to 2 × 107). PCR inhibitors were removed by extracting viral RNA (after virus concentration) using the RNeasy® mini kit with added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of enterovirus RNA allows large numbers of samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.

ABSTRACT The Epstein-Barr virus (EBV) genome can persist in dividing human B cells as multicopy circular episomes. Viral episomes replicate in synchrony with host cell DNA and are maintained at a relatively constant copy number for a long time. Only two viral elements, the replication origin OriP and the EBNA-1 protein, are required for the persistence of viral genomes during latency. EBNA-1 activates OriP during the S phase and may also contribute to the partition and/or retention of viral genomes during mitosis. Indeed, EBNA-1 has been shown to interact with mitotic chromatin. Moreover, viral genomes are noncovalently associated with metaphase chromosomes. This suggests that EBNA-1 may facilitate the anchorage of viral genomes on cellular chromosomes, thus ensuring proper partition and retention. In the present paper, we have investigated the chromosome-binding activity of EBV EBNA-1, herpesvirus papio (HVP) EBNA-1, and various derivatives of EBV EBNA-1, fused to a variant of the green fluorescent protein. The results show that binding to metaphase chromosomes is a common property of EBV and HVP EBNA-1. Further studies indicated that at least three independent domains (CBS-1, -2, and -3) mediate EBNA-1 binding to metaphase chromosomes. In agreement with the anchorage model, two of these domains mapped to a region that has been previously demonstrated to be required for the long-term persistence of OriP-containing plasmids.

Epstein-Barr virus has been associated to several forms of neoplasia including Burkitt's lymphoma, B lymphomas in immuno-compromised patients and undifferentiated nasopharyngeal carcinoma. Immunodepression, genetic and/or environmental factors and the expression of several viral genes (latent genes mainly) may contribute to these pathologies. In vitro, several latent proteins (EBNA 1, 2, 3A, 3C, 5 et LMP-1) directly or indirectly contribute to the initiation and maintenance of the transformation process, The role of these proteins is discussed in the present article.

Epstein-Barr Virus (EBV) is the prototype member of the human herpesvirus subfamily Gammaherpesviridae. EBV establishes a latent infection in lymphoïd cell and replicates in epithelial cells. Infection of individuals with EBV is widespread in all human populations, as shown by the high proportion of individual with specific antibodies in their serum. EBV is the etiological agent of the infectious mononucleosis and has been implicated in the pathogenesis of an increasing number of human malignancies, the best characterized being endemic Burkitt's lymphomas, nasopharyngeal carcinomas (NPC), and polyclonal lymphomas in immunocompromised patients. EBV infection in vivo is a complex mixture of latent, reactivated, transforming, or replicative type of infection. EBV infection is characterized by sporadic viral excretion in the oropharynx and persistent latent infection in the bone marrow and peripheral blood lymphocytes. EBV infection includes an intense immune response. During primary infection, the humoral response is primarily directed toward antigens of the lytic cycle, membrane antigen complex, early antigen (EA), viral capsid antigen (VCA) while the antibodies response to EBNAs is delayed. The cell mediated response controls the proliferation of EBV-infected lymphocytes through two classes of cytotoxic cells, namely, natural killer and T8 cytotoxic cells. Laboratory diagnosis of EBV infection is recently based upon molecular biology techniques which provides a useful tool for direct identification of EBV and may allow to better understand the role of the virus in the pathogenesis of EBV associated disorders.

Epstein-Barr Virus (EBV) is the prototype member of the human herpesvirus subfamily Gammaherpesviridae. EBV establishes a latent infection in lymphoïd cell and replicates in epithelial cells. Infection of individuals with EBV is widespread in all human populations, as shown by the high proportion of individual with specific antibodies in their serum. EBV is the etiological agent of the infectious mononucleosis and has been implicated in the pathogenesis of an increasing number of human malignancies, the best characterized being endemic Burkitt's lymphomas, nasopharyngeal carcinomas (NPC), and polyclonal lymphomas in immunocompromised patients. EBV infection in vivo is a complex mixture of latent, reactivated, transforming, or replicative type of infection. EBV infection is characterized by sporadic viral excretion in the oropharynx and persistent latent infection in the bone marrow and peripheral blood lymphocytes. EBV infection includes an intense immune response. During primary infection, the humoral response is primarily directed toward antigens of the lytic cycle, membrane antigen complex, early antigen (EA), viral capsid antigen (VCA) while the antibodies response to EBNAs is delayed. The cell mediated response controls the proliferation of EBV-infected lymphocytes through two classes of cytotoxic cells, namely, natural killer and T8 cytotoxic cells. Laboratory diagnosis of EBV infection is recently based upon molecular biology techniques which provides a useful tool for direct identification of EBV and may allow to better understand the role of the virus in the pathogenesis of EBV associated disorders.