First administration to humans of a monoclonal antibody cocktail against rabies virus: safety, tolerability, and neutralizing activity.

Immediate passive immune prophylaxis as part of rabies post-exposure prophylaxis (PEP) often cannot be provided due to limited availability of human or equine rabies immunoglobulin (HRIG and ERIG, respectively). We report first clinical data from two phase I studies evaluating a monoclonal antibody cocktail CL184 against rabies. The studies included healthy adult subjects in the USA and India and involved two parts. First, subjects received a single intramuscular dose of CL184 or placebo in a double blind, randomized, dose-escalation trial. Second, open-label CL184 (20IU/kg) was co-administered with rabies vaccine. Safety was the primary objective and rabies virus neutralizing activity (RVNA) was investigated as efficacy parameter. Pain at the CL184 injection site was reported by less than 40% of subjects; no fever or local induration, redness or swelling was observed. RVNA was detectable from day 1 to day 21 after a single dose of CL184 20 or 40IU/kg. All subjects had adequate (>0.5IU/mL) RVNA levels from day 14 onwards when combined with rabies vaccine. CL184 appears promising as an alternative to RIG in PEP.

A sensitive cell-based assay for the detection of residual infectious West Nile virus.

Ensuring complete viral inactivation is critical for the safety of vaccines based on an inactivated virus. Detection of residual infectious virus is dependent on sensitivity of the assay, sample volume analyzed and the absence of interference with viral infection. Here we describe the development and qualification of a sensitive cell-based assay for the detection of residual infectious West Nile Virus (WNV). The results of the assay are in good agreement with the assumption that at low concentrations the number of infectious units in relatively small samples follows a Poisson distribution. The assay can detect 1 infectious unit with a confidence of 99%, provides statistical controls for interference and can easily be scaled up to test large amounts of vaccine material. Furthermore, we show equivalence in sensitivity between the cell-based assay and an in vivo assay for detection of infectious WNV. Finally, the assay has been used for successful release testing of clinical lots of inactivated WNV vaccine. Given the principle and generic setup of the method we envision broad applicability to the detection of very low concentrations of infectious virus.

T cell expansions in lymph nodes and peripheral blood in HIV-1-infected individuals: effect of antiretroviral therapy.

OBJECTIVE: To evaluate dynamics in CD8 T cell expansions during highly active antiretroviral therapy (HAART). DESIGN: Various T cell subsets were isolated from blood and lymph nodes and analysed for T cell receptor (TCR) diversity. METHODS: TCR complementarity determining region 3 (CDR3) spectratyping and single-strand conformation polymorphism (SSCP) analyses were performed in combination with sequencing to assess clonality of the subsets. RESULTS: Strongly skewed CDR3 patterns in total CD8 cells and the CD8 subsets CD45RO+CD27+ and CD45RO-CD27+ showed substantial dynamics in dominant CDR3 sizes, resulting in relative improvement of CDR3 size diversity in the first months of therapy. During sustained treatment, TCR diversity changed only moderately. SSCP profiles confirmed oligoclonality of TCR CDR3 perturbations. Various dominant CDR3 sizes for CD4 and CD8 T cells present in lymph nodes, but not in peripheral blood mononuclear cells, before the start of therapy emerged in peripheral blood early during therapy. CONCLUSIONS: HAART induces substantial changes in CD8 TCR diversity, eventually resulting in improvement of the repertoire. Clonal expansions observed in lymph nodes before therapy were observed in peripheral blood after therapy, suggesting that recirculation of CD4 and CD8 T cells from lymph nodes contributes to the early T cell repopulation. Decreased immune activation and possibly naive T cell regeneration subsequently decreased clonal expansions and perturbations in the CD8 TCR repertoire.

Dysfunctional Epstein-Barr virus (EBV)-specific CD8(+) T lymphocytes and increased EBV load in HIV-1 infected individuals progressing to AIDS-related non-Hodgkin lymphoma.

Acquired immunodeficiency syndrome-related non-Hodgkin lymphomas (AIDS-NHL) are thought to arise because of loss of Epstein-Barr Virus (EBV)-specific cellular immunity. Here, an investigation was done to determine whether cellular immunity to EBV is lost because of physical loss or dysfunction of EBV-specific cytotoxic T cells. Data on EBV-specific cellular immunity were correlated with EBV load. For comparison, individuals who progressed to AIDS with opportunistic infections (AIDS-OI) and long-term asymptomatics (LTAs) were studied. The number of virus-specific T cells was detected using tetrameric HLA-EBV-peptide complexes; function of these EBV-specific T cells was determined using the interferon-gamma (IFN-gamma) Elispot assay. It was observed that EBV-specific CD8(+) T cells were present in normal numbers in human immunodeficiency virus (HIV)-infected individuals. However, their functional capacity was decreased compared with HIV(-) individuals. In AIDS-NHL patients, EBV-specific T cells were not physically lost in the course of HIV-1 infection but showed progressive loss of their capability to produce IFN-gamma in response to EBV peptides. This loss of function correlated with lower CD4(+) T-cell numbers and was accompanied by increasing EBV load. In HIV-1-infected LTA individuals, in whom CD4(+) T-cell numbers were maintained, and progressors to AIDS-OI, IFN-gamma-producing EBV-specific T cells were stable and EBV load remained stable or decreased in the course of HIV infection, suggestive of immune control. Our data indicate that functional loss of EBV-specific CD8(+) T cells with a concomitant increase in EBV load may play a role in the pathogenesis of AIDS-NHL.

High viral burden in the presence of major HIV-specific CD8(+) T cell expansions: evidence for impaired CTL effector function.

To investigate the effect of HIV-specific CD8(+) T cells on viral plasma load and disease progression, we enumerated HLA-A2-, B8- and B57-restricted CD8(+) T cells directed against several HIV epitopes in a total of 54 patients by the use of tetrameric HLA-peptide complexes. In patients with high CD4(+) T cell numbers, HIV-specific tetramer(+) cells inversely correlated with viral load. Patients with CD4(+) T cell numbers below 400/microl blood, however, carried high viral load despite frequently having high tetramer(+) T cell numbers. This lack of correlation between viral load and tetramer(+) cells did not result from viral escape variants, as in only 4 of 13 patients, low frequencies of viruses with mutated epitopes were observed. In 15 patients we measured CD8(+) T cell antigen responsiveness to HIV peptide stimulation in vitro. FACS analyses showed differential IFN-gamma production of the tetramer(+) cells, and this proportion of IFN-gamma-producing tetramer(+) cells correlated with AIDS-free survival and with T cell maturation to the CD27(-) effector stage. These data show that most HIV-infected patients have sustained HIV-specific T cell expansions but many of these cells seem not to be functional, leaving the patient with high numbers of non-functional virus-specific CD8(+) T cells in the face of high viral burden.

Longitudinal phenotypic analysis of human immunodeficiency virus type 1-specific cytotoxic T lymphocytes: correlation with disease progression.

Few studies have examined longitudinal changes in human immunodeficiency virus type 1 (HIV)-specific cytotoxic T lymphocytes (CTL). To more closely define the natural history of HIV-specific CTL, we used HLA-peptide tetrameric complexes to study the longitudinal CD8(+) T-cell response evolution in 16 A*0201-positive untreated individuals followed clinically for up to 14 years. As early as 1 to 2 years after seroconversion, we found a significant association between high frequencies of A*0201-restricted p17(Gag/Pol) tetramer-binding cells and slower disease progression (P < 0.01). We observed that responses could remain stable over many months, but any longitudinal changes that occurred were typically accompanied by reciprocal changes in RNA viral load. Phenotypic analysis with markers CD45RO, CD45RA, and CD27 identified distinct subsets of antigen-specific cells and the preferential loss of CD27(+) CD45RO(+) cells during periods of rapid decline in the frequency of tetramer-binding cells. In addition we were unable to confirm previous studies showing a consistent selective loss of HIV-specific cells in the context of sustained Epstein-Barr virus-specific cell frequencies. Overall, these data support a role of HIV-specific CTL in the control of disease progression and suggest that the ultimate loss of such CTL may be preferentially from the CD27(+) CD45RO(+) subset.

Evidence that human CD8+CD45RA+CD27- cells are induced by antigen and evolve through extensive rounds of division.

We recently showed that circulating human CD8(+) effector cells have a CD45RA+CD27(-) membrane phenotype. In itself this phenotype appeared to pose a paradox: CD45RA, a marker expressed by unprimed cells, combined with absence of CD27, characteristic for chronically stimulated T cells. To investigate whether differentiation towards the CD45RA+CD27(-) phenotype is dependent on antigenic stimulation and involves cellular division, TCR Vbeta usage and telomeric restriction fragment (TRF) length were analyzed within distinct peripheral blood CD8(+) subsets. FACS analysis showed that the TCR Vbeta repertoire of CD8(+)CD45RA+CD27(-) cells differed significantly from that of unprimed CD8(+)CD45RA+CD27(+) cells. Moreover, in two out of six individuals large expansions of particular Vbeta families were observed in the CD8(+)CD45RA+CD27(-) subset. CDR3 spectrotyping and single-strand confirmation analysis revealed that within the CD8(+)CD45RA+CD27(-) population most of the 22 tested Vbeta families were dominated by oligoclonal expansions. The mean TRF length was found to be 2.3+/-1.0 kb shorter in the CD8(+)CD45RA+CD27(-) subset compared with the unprimed CD8(+)CD45RA+CD27(+) population, but did not differ substantially from that of memory type, CD8(+)CD45RA-CD27(+) T cells. These findings indicate that the CD8(+)CD45RA+CD27(-) cytotoxic effector population consists of antigen-induced, clonally expanded cells and confirm that the expression of CD45RA is not a strict marker of antigen non-experienced T cells.

Interleukin 12 administration enhances Th1 activity but delays recovery from influenza A virus infection in mice.

Interleukin 12 (IL-12) directs the differentiation of undifferentiated T helper (Th0) cells to T helper type 1 (Th1) cells and induces a cell-mediated immune response. To evaluate the effect of IL-12 on the course of influenza A virus infection, BALB/c mice were administered a daily intraperitoneal dose of 1000 ng of IL-12 or saline on days -1 to +4 for a total of six treatments. The treatment generally enhanced Th1-mediated responses. IFNgamma lung concentrations were 1193 +/- 275 pg/100 microl in controls and 3693 +/- 745 pg/100 microl in IL-12-treated mice at day 5. IFNgamma levels were undetectable at day 13 in controls and 1335 +/- 220 pg/100 microl in IL-12-treated mice. Cytokine production was also assessed at the single-cell level for mediastinal lymph nodes. IL-12 treatment increased the number of IL-2- and IFNgamma-producing cells and decreased the number of IL-4- and IL-10-producing cells. IL-12 treatment decreased the anti-influenza antibody response, especially anti-influenza IgG1 antibody resulting in an increased IgG2a/IgG1 ratio. Primary pulmonary CTL activity on day 5 was low for both groups (10% specific lysis). Secondary CTL activity at day 11 was higher for control mice than for IL-12-treated mice on day 11 (44 versus 34%), but not on day 13. Despite this overall enhancement of Th1-mediated immune functions, the IL-12 treatment increased severity of the disease. Following infection, control and IL-12-treated mice decreased their body weight to approximately 75% of their initial weight. After day 5, the control mice started to recover, while IL-12-treated mice did not begin recovering until day 9. Pulmonary viral titers were 1.6 +/- 0.3 TCID50 in controls at day 5 compared to 2.4 +/- 0.3 for IL-12-treated mice (P < 0.01). In addition, control mice had significantly less severe inflammation and damage on histologic examination. Serum TNFalpha concentrations, undetectable in control mice, were elevated by IL-12 treatment up to 80 pg/ml at day 5 and decreased to zero at day 13. It is concluded that IL-12 administration to influenza-infected mice induces a switch from a Th2- to a Th1-mediated response, but inhibits recovery probably through induction of TNFalpha.

Diversity of the T-cell receptor BV repertoire in HIV-1-infected patients reflects the biphasic CD4+ T-cell repopulation kinetics during highly active antiretroviral therapy.

OBJECTIVES: Highly active antiretroviral therapy (HAART) induces a decline in viral load and a biphasic increase in peripheral blood CD4+ T-cell counts in HIV-infected patients. To evaluate the effect of HAART on T-cell receptor (TCR) diversity of repopulating naive and memory CD4+ T cells, complementarity determining region 3 (CDR3) spectratyping was performed. DESIGN: For four patients treated with HAART, CD45RO+ (memory) and CD45RA+ (naive) CD4+ T cells were isolated from peripheral blood leukocyte samples obtained 1 week before, 1-2 months after, and 9-11 months after start of treatment. METHODS: CDR3 regions were amplified by TCR-BV-specific nested PCR from CD4+ T-cell subsets. CDR3 size distributions and single-strand conformation polymorphism profiles were compared as an indication for TCR diversity. RESULTS: Increasing blood CD4+ T-cell counts during the first 2 months of treatment coincided with increased perturbation of CDR3 patterns in CD4+ T-cell subsets, suggesting an early oligoclonal repopulation. At later timepoints, CDR3 size diversity increased when T-cell counts did not substantially decrease. Memory and naive CD4+ T cells generally showed comparable levels of perturbation. CONCLUSION: Diversity of the TCR repertoire reflected biphasic T-cell repopulation during HAART, compatible with initial redistribution and later CD4+ T-cell production. Sustained elevation of T-cell counts will in principle result in restoration of TCR diversity.