Persistent numbers of tetramer+ CD8(+) T cells, but loss of interferon-gamma+ HIV-specific T cells during progression to AIDS

Although CD8(+) T cells initially suppress human immunodeficiency virus (HIV) replication, cytotoxic T-cell precursor frequencies eventually decline and fail to prevent disease progression. In a longitudinal study including 16 individuals infected with HIV-1, we studied both the number and function of HIV-specific CD8(+) T cells by comparing HLA-peptide tetramer staining and peptide-induced interferon-gamma (IFN-gamma) production. Numbers of IFN-gamma-producing T cells declined during progression to acquired immunodeficiency syndrome (AIDS), whereas the number of tetramer+ T cells in many individuals persisted at high frequencies. Loss of IFN-gamma-producing T cells correlated with declining CD4(+) T-cell counts, consistent with the need of CD4(+) T-cell help in maintaining adequate CD8(+) T-cell function. These data indicate that the loss of HIV-specific CD8(+) T-cell activity is not due to physical depletion, but is mainly due to progressively impaired function of HIV-specific CD8(+) T cell

Characterization of virus-specific CD8(+) effector T cells in the course of HIV-1 infection: longitudinal analyses in slow and rapid progressors

Studies in humans have provided evidence that CD8(+) T cells exhibit distinct phenotypical and functional properties dependent on virus specificity. It is not known how these T-cell phenotypes develop over the course of infection. Dynamics and properties of T cells specific for human immunodeficiency virus (HIV), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) in HIV infection were investigated in relation to viral load. In rapid progressors, HIV-specific CD8(+) T cells were less differentiated early in infection and did not develop a more differentiated phenotype. In slow progressors, perforin expression of HIV-specific CD8(+) T cells slightly increased over time. HIV and EBV loads were detectable in all individuals, while CMV load could not be detected. Thus, in individuals with progressive HIV infection, HIV-specific T cells are less differentiated already early in infection. This apparent block in differentiation may be partly caused by chronic viremia or lack of CD4(+) T-cell help. (C) 2004 Elsevier Inc. All rights reserve

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

Quantifying Adenovirus-Neutralizing Antibodies by Luciferase Transgene Detection: Addressing Preexisting Immunity to Vaccine and Gene Therapy Vectors

The presence of various levels of anti-adenovirus serotype 5 (Ad5)-neutralizing antibodies in humans is thought to contribute to the inconsistent clinical results obtained so far in diverse gene transfer and vaccination studies and might preclude universal dosing with recombinant Ad5. Prescreening of individuals eligible for Ad5 or alternative serotype treatment and subsequently tailoring the vector dose might aid in ensuring the consistency of clinical parameters. For this purpose, a qualified Ad neutralization assay is required. Here we have tested the different protocols used to date to determine anti-Ad neutralizing activity. Based on simplicity, speed, high throughput, sensitivity, and robustness, we propose a qualified assay in which Ad neutralization is monitored by luciferase reporter gene expression

Validation of the rapid fluorescent focus inhibition test (RFFIT) for rabies virus neutralizing antibodies in clinical samples

Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two antibodies are developed as replacements of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) in postexposure prophylaxis (PEP). The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay which is usually performed to determine the biological potency of a vaccine and to measure the levels of protection against rabies in humans and animals. In order to confirm the suitability of this assay as a pharmacodynamic assay, we conducted a validation using both HRIG- and CL184-spiked serum samples and sera from vaccinated donors. The validation results met all analytical acceptance criteria and showed that HRIG and CL184 serum concentrations can be compared. Stability experiments showed that serum samples were stable in various suboptimal conditions but that rabies virus should be handled swiftly once thawed. We concluded that the assay is suitable for the measurement of polyclonal and monoclonal rabies neutralizing antibodies in clinical serum samples

A fit-for-purpose strategy for the risk-based immunogenicity testing of biotherapeutics - a European industry perspective

There is much debate in the pharmaceutical industry on how to translate the current guidelines on immunogenicity testing for biotherapeutics into a testing strategy that suits the specific requirements of individual drug candidates. In this paper, member companies from the European immunogenicity platform (EIP) present a consensus view on the essential requirements for immunogenicity testing of a biotherapeutic throughout the various phases of drug development, to ensure patient safety and to enable successful market entry. Our aim is to open the debate and provoke discussion on this important topic which is unique to biotherapeutic drug development. The scope of this paper is limited to aspects relevant to biotherapeutic drug development and does not include fundamental academic studies of immunogenicity. Here, we propose two pre-defined testing strategies for the detection and characterization of anti-drug antibody (ADA) responses where the different strategies are based on the phase of development for a biotherapeutic, a. without (category 1) and b. with (category 2) the expected potential to elicit ADA mediated severe clinical consequences. The harm of a potential ADA response determines which of the two testing strategies is adopted. The scientific rationale on which the "case-by-case" approach advocated in white papers and guidance documents may be translated for each individual drug development program is provided and, underpins the recommendations made here

Adenovirus types 5 and 35 seroprevalence in AIDS risk groups supports type 35 as a vaccine vector

The seroprevalence of adenovirus types 5 (Ad5) and 35 (Ad35) was investigated in patients at risk of AIDS. The seroprevalence of Ad5 was higher than Ad35 in HIV-infected patients from the Netherlands (60% versus 7%) and sub-Saharan Africa (90% versus 20%). The seroprevalence was similar among HIV-infected and uninfected individuals, and remained constant during progression to AIDS. Ad35 is less prone to neutralization than Ad5, encouraging the further development of Ad35 for vaccination against HI

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