[Lemierre syndrome variant: Hepatic abscesses and hepatic venous thrombosis due to Fusobacterium nucleatum septicemia]. / Un variant du syndrome de Lemierre: abcès hépatiques et thrombose veineuse sus-hépatique secondaire à une septicémie à Fusobacterium nucleatum.

INTRODUCTION: Like Fusobacterium necrophorum, Fusobacterium nucleatum is capable causing Lemierre's syndrome. Various locations of venous thrombosis have been described associated with Fusobacterium sp. septicemia. EXEGESIS: We describe a 43-year old alcoholic patient with F.nucleatum septicemia complicated with hepatic abscesses, middle hepatic venous thrombosis, osteomyelitis and infiltrative pneumonia. A pancreatic prosthesis was the only potentially identified infectious entrance. CONCLUSION: Our patient showed an alternative presentation of Lemierre's syndrome, a "digestive variant". To the best of our knowledge, this is the first report of Fusobacterium septicemia associated with hepatic venous thrombosis. This report is close to the cases of portal thrombosis and opens the clinical sphere of the lemierre's syndrome, whose incidence is increasing.

Markov modelling of changes in HIV-specific cytotoxic T-lymphocyte responses with time in untreated HIV-1 infected patients.

HIV-specific cytotoxic CD8(+) T-lymphocytes (CTL) appear to be the cornerstone of the immune response to HIV infection. Recent studies show that CTL activity reflects patients' anti-HIV immune status and slows disease progression. However, the dynamics of the diversity of this response also appears as a key parameter for immune control but the dynamics of this diversity is largely undocumented. We modelled changes in CTL responses against the seven principal HIV proteins over time. We also studied the influence of plasma viral load on temporal changes in HIV protein recognition by memory CTL. The generic model we developed is based on a continuous time homogeneous Markov process with reversible states. Those states are defined by the number of proteins recognized by memory CTL in a given patient at a given time. This approach was developed within a Bayesian framework. Full Bayesian inference is implemented using Markov chain Monte Carlo simulations (MCMC). The Gibbs sampling algorithm was used to estimate the marginal posterior distributions of the transition intensities between stages of CTL responses. We applied our model to data of 152 HIV-infected patients included in the IMMUNOCO cohort. The model suggested that the diversity of HIV protein recognition by memory CTL in treatment-naive patients decreases as the disease progresses. Namely, the loss of T cytotoxic responses is globally faster than their acquisition. Indeed, these patients' T cytotoxic responses were characterized by marked individual turnover and a gradual loss of multiple protein recognition over time, this loss accelerating as viral load increased.

Comparison of subcutaneous and intravenous interleukin-2 in asymptomatic HIV-1 infection: a randomised controlled trial. ANRS 048 study group.

BACKGROUND: Intermittent interleukin-2 therapy for HIV-1 by continuous intravenous infusion leads to sustained increase of CD4 T cells. This method of administration is, however, inconvenient and has limiting toxic effects. We did a randomised study to compare safety and efficacy of antiviral treatment alone or combined with various interleukin-2 regimens in HIV-1-infected patients. METHODS: 94 symptom-free patients, naïve to antiretroviral treatment, with CD4-T-cell counts of 250-550 cells/microL at baseline were randomly assigned zidovudine and didanosine alone (n=26) or combined with interleukin-2 administered intravenously (12 million IU/day, n=22) or subcutaneously (3 million IU/m2 twice daily, n=24) for 5 days, or were given polyethylene-glycol-modified (PEG) interleukin-2 (2 million IU/m2 intravenous bolus, n=22) administered every 2 months from week 2 to week 50 (seven cycles). Safety and immunological and virological results were monitored until week 56. FINDINGS: CD4-T-cell count increased to higher than baseline by a mean of 564 cells/microL (subcutaneous group), 676 cells/microL (intravenous group), 105 cells/microL (PEG group), and 55 cells/microL (antiretroviral-therapy group, p=0.0001). 68% and 77% of patients in the subcutaneous and intravenous groups, respectively, achieved an 80% increase of CD4 T cells (p<0.001). In these two groups, 50% of patients restored a CD4/CD8-T-cell ratio of more than 1. The groups did not differ significantly for changes in plasma HIV-1 RNA loads throughout the study. The duration of common side-effects of interleukin-2 was shorter in the subcutaneous group, which enabled outpatient treatment. Naïve and memory CD4 T cells, CD28 expression on CD4 and CD8 T cells, and restoration of in-vitro proliferative response to mitogens and recall antigens increased in the intravenous and subcutaneous groups. INTERPRETATION: Subcutaneous interleukin-2 is a convenient regimen that, as well as intravenous therapy, improves immunological function in HIV-1-infected patients receiving two nucleosides. Larger studies are needed to show whether immunological improvements translate into clinical benefit.

Cytotoxic T-cell responses to HIV-1 reverse transcriptase, integrase and protease.

OBJECTIVES: To determine immunodominant regions and new epitopes for cytotoxic T cells (CTL) directed against the HIV-1 pol products reverse transcriptase (RT), integrase and protease in a large cohort of patients at different stages of disease. DESIGN AND METHODS: Cross-sectional analysis of 98 patients from the French IMMUNOCO cohort (CD4 counts: 125-1050 x 10(6) cells/l), monitored for CTL recognition of HIV-1 pol products using recombinant vaccinia virus constructs and synthetic peptides. RESULTS: Memory CTL responses against HIV-1 pol products were detected in 78% of all patients whatever the stage of disease. RT was more immunogenic (81%, 30 out of 37 patients) than integrase and protease (51% and 24%, respectively). CTL recognition of RT was more frequent against Pol amino acids 310-460 (61%, 11 out of 18 patients) than against the other three portions (Pol 168-310, Pol 450-600, Pol 590-728) in patients with CD4 counts > 400 x 10(6)/l, whereas in patients at advanced stages no prominent differences were observed. Two new clusters of antigenic regions were found in the NH2 segment: three epitopes between amino-acids Pol 200 and 217 and four epitopes between amino-acids Pol 346 and 387, using five different HLA-restricting elements. A new cluster of three conserved epitopes was found in the COOH segment of RT. CONCLUSIONS: This study shows that memory CTL responses against HIV-1 RT, integrase and protease are detectable in most patients at different stages of disease. The capacity of CTL to recognize simultaneously clusters of epitopes may become important for the immune control to reinforce antiretroviral drug efficiency.

Evolution of cytotoxic T lymphocyte responses to human immunodeficiency virus type 1 in patients with symptomatic primary infection receiving antiretroviral triple therapy.

The impact of highly active antiretroviral treatment (HAART) on anti-human immunodeficiency virus (HIV) cytotoxic T lymphocytes (CTL) was studied in 17 patients with recent symptomatic HIV-1 primary infection receiving triple combination therapy. Anti-HIV CTL were initially detected in 15 patients. In 6, CTL disappeared rapidly and persistently after initiation of therapy. Most of them had a rapid and sustained decrease in plasma HIV RNA to undetectable levels. Conversely, in 6 other patients, CTL remained detectable, which was associated with a less efficient control of viral replication. In 3 others, CTL disappeared only transiently, without clear correlation with the virologic profile. Altogether, despite individual variations, there was a positive correlation between viral replication and anti-HIV-1 cytotoxicity in most subjects, suggesting that the persistence of viral antigens is the main determinant for the maintenance of CTL activity. This raises the question of the potential benefit of anti-HIV CTL induction by immunotherapy in acute seroconverters treated by HAART.

Adjuvant is required when using Env lipopeptide construct to induce HIV type 1-specific neutralizing antibody responses in mice in vivo.

Extensive immunological studies on HIV-1 infection, the causative agent of AIDS in humans, have led to the conclusion that efficient protection against this infection should require early elicitation of neutralizing antibodies as well as cellular immune and particularly cytotoxic T lymphocyte responses. The use of synthetic peptides modified at one end by introduction of a lipidic tail is now well known to be an effective means of eliciting virus-specific cytotoxic T lymphocyte responses in vivo, both in mouse and humans. To ascertain that such a strategy can be used for vaccinal purposes, particularly against HIV-1 infection, it remains to be determined whether these molecules can also act as effective inducers of antibody responses, most of all of the neutralizing type. The present study set out to address this question by using a synthetic HIV-1 ENV lipopeptide construct, previously identified as a potent immunogen for in vivo induction of ENV-specific CTL responses in BALB/c mice. We first showed that V3 peptide-specific antibodies were effectively induced by the lipopeptide construct. However, we provided evidence that the biological activity of these antibodies, i.e., their ability to neutralize HIV-1 infectivity in vitro, was strongly influenced by the immunizing conditions and protocol, in that only those antibodies generated by the use of adjuvanted lipopeptide formulations were effective. Albeit at a slightly lower efficacy than by the intraperitoneal route, neutralizing antibodies could also be induced using the subcutaneous route. With the prospect of a human peptide vaccine in mind, we then studied the properties of different known or possibly clinically relevant adjuvants. We found that alum, the only relevant adjuvant for human use, not only provides inefficient help to the lipopeptide construct in generating neutralizing antibodies, but tends to have deleterious effects on the ability of the construct to induce CTL responses. The only protocol that gave satisfactory results in terms of the magnitude of the neutralizing antibody responses was a mineral oil-based lipopeptide formulation. When induced under those conditions, strong neutralizing activities were still present up to 8 months after the last injection.

Study of the immunogenicity of different recombinant Mengo viruses expressing HIV1 and SIV epitopes.

Recombinant Mengo viruses expressing heterologous genes have proven to be safe and immunogenic in both mice and primates, and to be able to induce both humoral and cellular immune responses (Altmeyer et al., 1995, 1996). Several recombinant Mengo viruses expressing either a large region (aa 65-206) of the HIV1 nef gene product, or cytotoxic T lymphocyte (CTL) epitopic regions from the SIV Gag (aa 182-190), Nef (aa 155-178) and Pol (aa 587-601) gene products were engineered. The heterologous antigens were expressed either as fusion proteins with the Mengo virus leader (L) protein, or in cleaved form through autocatalytic cleavage by the foot-and-mouth disease virus 2A protein. Rhesus macaques and BALB/c mice inoculated with the Mengo virus SIV recombinants failed to develop CTL responses against the SIV gene products, while one of the HIV-Nef recombinants induced a weak CTL response in mice directed to an HIV1 Nef peptide spanning positions 182-198. In contrast, BALB/c mice immunized with vaccinia virus recombinants expressing HIV1 Nef developed a strong CTL response to the 182-198 peptide and also responded to a second peptide spanning positions 73-81. These results indicate that Mengo virus recombinants expressing HIV1 Nef and SIV CTL epitopes are weak immunogens. One of the fusion recombinants expressing SIV CTL epitopes failed to infect macaques even when used at high doses, while the recombinant expressing HIV1 Nef as a fusion protein failed to infect BALB/c mice. These results demonstrate that the expression of certain heterologous sequences as fusion proteins with L can result in the loss of the ability of the recombinant to infect normally susceptible animals.

Cross-reactions between the cytotoxic T-lymphocyte responses of human immunodeficiency virus-infected African and European patients.

The great variability of protein sequences from human immunodeficiency virus (HIV) type 1 (HIV-1) isolates represents a major obstacle to the development of an effective vaccine against this virus. The surface protein (Env), which is the predominant target of neutralizing antibodies, is particularly variable. Here we examine the impact of variability among different HIV-1 subtypes (clades) on cytotoxic T-lymphocyte (CTL) activities, the other major component of the antiviral immune response. CTLs are produced not only against Env but also against other structural proteins, as well as some regulatory proteins. The genetic subtypes of HIV-1 were determined for Env and Gag from several patients infected either in France or in Africa. The cross-reactivities of the CTLs were tested with target cells expressing selected proteins from HIV-1 isolates of clade A or B or from HIV type 2 isolates. All African patients were infected with viruses belonging to clade A for Env and for Gag, except for one patient who was infected with a clade A Env-clade G Gag recombinant virus. All patients infected in France were infected with clade B viruses. The CTL responses obtained from all the African and all the French individuals tested showed frequent cross-reactions with proteins of the heterologous clade. Epitopes conserved between the viruses of clades A and B appeared especially frequent in Gag p24, Gag p18, integrase, and the central region of Nef. Cross-reactivity also existed among Gag epitopes of clades A, B, and G, as shown by the results for the patient infected with the clade A Env-clade G Gag recombinant virus. These results show that CTLs raised against viral antigens from different clades are able to cross-react, emphasizing the possibility of obtaining cross-immunizations for this part of the immune response in vaccinated individuals.

In vivo longitudinal analysis of a dominant TCR repertoire selected in human response to influenza virus.

Recent studies have demonstrated biased usage of TCR V beta 17 and a high degree of diversity in J beta usage within the influenza virus matrix epitope (M.58-66)-specific CTL response. In contrast, in the course of a study on the cellular response to influenza A virus, we found preferential usage of V beta 17-J beta 2.2 rearrangement in an individual with an unexpectedly high number of CTL precursors (CTLp). We took advantage of such situation to study the longitudinal repertoire of the CD8+ T cell precursors. By limiting dilution analysis combined with the use of a clonotypic primer corresponding to the CDR3 region of this matrix-specific TCR V beta chain, the influenza-specific CTLp were shown to be stable for a period of 6 years. Overall, our results show that virus-specific CTLp can be directly monitored in vivo by molecular fingerprinting without in vitro restimulation. These findings might be extremely important for evaluation of the specific immune response to a given human pathogen.

Human peptide transporter deficiency: importance of HLA-B in the presentation of TAP-independent EBV antigens.

Two siblings with a peptide TAP deficiency were recently described. Despite poor cell surface expression of HLA class I molecules, these patients were not unusually susceptible to viral infections. The majority of the cell surface-expressed class I molecules were HLA-B products as assessed by cytofluorometry and biochemical analysis. Analysis of two peptides eluted from the class I molecules expressed by TAP-deficient EBV B lymphoblastoid cell lines indicated that both were derived from cytosolic proteins and presented by HLA-B molecules. Peripheral alphabeta CD8+ T cells were present and their TCR repertoire was polyclonal. Most of the alphabeta CD8+ T cell clones studied (21 of 22) were nonreactive against cells expressing normal levels of the same HLA alleles as those of the TAP-deficient patients. However, it was possible to isolate one cytotoxic CD8+ alphabeta T cell clone recognizing the EBV protein LMP2 presented by HLA-B molecules on TAP-deficient cells. These observations suggest that in the TAP-deficient patients, CD8+ alphabeta T cells could mature and be recruited in immune responses to mediate HLA class I-restricted cytotoxic defense against viral infections. They also strengthen the physiologic importance of a TAP-independent processing pathway of the LMP2 protein, which was previously shown to contain several other TAP-independent epitopes.

Influence of strong CD4 epitope on long-term virus-specific cytotoxic T cell responses induced in vivo with peptides.

Free unmodified peptides are poor immunogens for cytotoxic T lymphocytes (CTL) in mice, unless they are injected with adjuvant. Although it is generally accepted that CD4+ Th cells are essential for CTL priming with peptides, it is not clear how long sequences devoid of any CD4 epitope, or strict CD8 epitopic sequences too short to be presented in a MHC class II-restricted fashion, can generate such responses. We thus have examined the extent to which the immunization protocol affects the need for a CD4 epitope. Since peptides are potentially important for vaccination, we also examined the duration of the CTL responses using a set of peptides that contained a CD4 epitope, or a CD8 epitope, or both epitopes in the same sequence, and compared immunization protocols previously found to induce CTL responses with peptides. The in vivo injection protocol had a marked effect, since the same CD8 sequence could generate a CTL response in a Th-dependent or Th-independent fashion, depending on the protocol used. The T cell help provided by natural CD4-CD8 sequences was inefficient in Th-dependent CTL priming and CTL generation required help from a stronger exogenous CD4 peptide. The peptides could be injected either as a single tandem CD8-CD4 peptide or as a mixture of two separate peptides. Th-independent CTL responses primed in other conditions proved to be as strong as Th-dependent responses, at least when the animals were killed shortly after the last injection. However, only CTL responses generated together with specific Th cells persisted for several months. Moreover, the efficacy of CTL persistence seemed to be correlated with the strength of the CD4 epitope for priming. This has important implications for the design of peptide vaccines.

Delayed virus-specific CD8+ cytotoxic T lymphocyte activity in an HIV-infected individual with high CD4+ cell counts: correlations with various parameters of disease progression.

This 4-year longitudinal study monitored the temporal association between the HIV-specific cytotoxic T lymphocyte (CTL) response and the control viremia in an individual infected with human immunodeficiency virus type (HIV-1). At the beginning of the study, this asymptomatic individual with a high CD4+ cell count showed no HIV-specific cytotoxic activity after polyclonal in vitro restimulation with autologous PHA-blasts, unlike most HIV-seropositive individuals. Anti-HIV CTLs were detected only in the last year of the study, both after in vitro restimulation and directly ex vivo. This was correlated with the inversion of the CD4+/CD8+ ratio, essentially due to increased numbers of CD8+CD28- T lymphocytes. The HIV-specific cytolytic activity was mediated by this CD28+CD28- subpopulation. The amount of HIV-1 provirus in peripheral blood mononuclear cells (PBMCs) did not change during the study, but the HIV RNA in plasma increased and virus was isolated from PBMCs only at the time when HIV-specific CTL activity was detected. This suggests overall that the HIV-1 replication was low in this individual, with a transient increase that could have reached the threshold for CTL reactivation, and was perhaps controlled thereby.

Comparative efficiency of simple lipopeptide constructs for in vivo induction of virus-specific CTL.

We have previously shown that virus-specific CTL responses can be elicited in vivo by injecting, without adjuvant, 12-40 amino acid-long peptides, modified in C-terminal position by a simple lipidic amino acid. In this paper, we have studied the chemical accessibility, and the ability to induce in mice a CTL response, of a series of lipopeptides derived from the HIV-1 env (312-327) or (302-335) sequences. We showed that a single modification of these peptides by a lipidic amino acid, preferably in C-terminal position, results in the ability to reproducibly induce, without adjuvant, a relevant CTL response. No clear discrimination appeared concerning the nature of the lipidic modification. Our findings indicate that modification of a relatively long peptide by a N epsilon-palmitoyl-L-Lysylamide can be achieved by conventional methods of synthesis and characterization, offering the possibility to develop low-cost synthetic vaccines in models in which the CTL component is of importance.

Predominant involvement of CD8+CD28- lymphocytes in human immunodeficiency virus-specific cytotoxic activity.

Distinct functional CD8+ T-cell populations have been observed during human immunodeficiency virus (HIV) infection. One of these functions is the inhibition of viral replication by a noncytotoxic mechanism, which was shown to be mediated by the CD8+CD28+ subpopulation. On the other hand, CD8+ T cells exert an HIV-specific cytotoxic activity. The present study shows that CD8+CD28- lymphocytes display this HIV-specific cytotoxic activity, which is detectable immediately after the cells are purified from peripheral blood. The CD28- population is also able to proliferate and to retain its cytotoxic activity after in vitro restimulation with autologous blast cells. Finally, HIV-specific cytotoxic T cells can be obtained in vitro from the CD8+CD28+ population.

Long-lasting anti-viral cytotoxic T lymphocytes induced in vivo with chimeric-multirestricted lipopeptides.

Cytotoxic T lymphocytes (CTL) play a major role in protective immunity against viral diseases. However, the antigenic formulations that can be used in vaccinations able to generate virus-specific CTL responses in vivo have yet to be defined. We have previously shown that HIV-1-specific CTL can be elicited in mice by injecting without adjuvant a synthetic peptide of the envelope glycoprotein that has been modified by the addition of a simple lipid tail to the end of the sequence (lipopeptide). The present study set out to address the question of whether such immunogens may be appropriate for preparing a human synthetic vaccine. We first showed that CTL were effectively induced by lipopeptides when given s.c. or i.p. We evidenced that the in vivo induction required stimulation of a concomitant specific T helper cell response, implying the presence of at least one CD4 epitope in the synthetic sequence used. Bearing in mind the particular properties that would be required in a prospective human peptide vaccine, we conceived a strategy in which a virus-specific CTL response could be generated in mice of different haplotypes using a single lipopeptide. We therefore tested a lipopeptide construct that integrated a synthetic sequence having three colinear epitopes of the influenza virus nucleoprotein, each restricted to a different H-2 haplotype. We found that a CTL response could be elicited to all three epitopes of this chimeric multirestricted lipopeptide construct. Finally, we have attempted to estimate the duration of the responses; strong CTL activities were still present up to six months after the last injection. These findings indicate that this approach may be suitable for developing a synthetic vaccine for human use.

Different patterns of HIV-1-specific cytotoxic T-lymphocyte activity after primary infection.

OBJECTIVE: To analyse the HIV-1-specific cytotoxic T-lymphocyte (CTL) responses of nine HIV-seropositive subjects in relation with primary infection. METHODS: Anti-HIV CTL were generated by in vitro stimulation of peripheral mononuclear cells obtained from HIV-seropositive donors at various times after primary infection. They were tested against several structural or regulatory HIV-1 proteins, using autologous target cells infected with recombinant vaccinia viruses expressing one of the HIV-1LAI proteins. RESULTS: An important CTL activity was found during the first month following seroconversion only in those donors who showed clinical symptoms during primary infection. The temporal evolution of this response differed for each subject; one remained a non-responder even 30 months after seroconversion. The structural proteins were recognized particularly early, while the antigenicity of regulatory proteins appeared later. CONCLUSION: Different patterns of HIV-specific CTL response can be observed after primary infection. The evolution of infection in these different HIV-seropositive subjects will be particularly interesting to analyse.