Eva Halapi

Neutralizing antibodies and viral characteristics in mother-to-child transmission of HIV-1.

ObjectiveTo determine viral characteristics and the protective effect of virus neutralizing antibodies in mother-to-child transmission of HIV-1. Molecular studiesTen HIV-1-infected mother-child pairs were sampled within 4 months of delivery. Variable region 3 of the viral envelope was amplified by nested polymerase chain reaction and sequenced, directly and/or after cloning, by solid-phase DNA sequencing. The amino acid sequence of variable region 3 from all 10 children was homogeneous, whereas the mothers showed varying degrees of heterogeneity. Apparently, selection of an HIV-1 variant occurs either at transmission or during initial virus replication in the infected child. No characteristic molecular features of the transmitted virus were identified. Biological studiesVirus isolates from 13 mother-child pairs were characterized for replicative capacity in a variety of cell lines. Eight mothers from whom a virus with a slow/low replicative pattern was isolated transmitted the slow/low virus to their children, whereas mothers with a rapid/high virus transmitted either a rapid/high or a slow/low virus (two cases each). This indicates that viruses with rapid/high replicative capacity do not have a selective advantage during transmission. Virus neutralizingSera from 20 mothers were characterized for the ability to neutralize their own virus (autologous neutralization) and virus from other mothers (heterologous neutralization). The results showed that non-transmitting mothers had neutralizing antibodies against autologous virus more frequently than transmitting mothers. In addition, all mothers with autologous neutralizing antibodies also neutralized at least two heterologous primary isolates. This indicates that a broad neutralizing antibody response may be linked to a lower risk of mother-to-child transmission. ConclusionOn the basis of the variable region 3 loop sequence, HIV-1-infected infants harbour homogenous virus populations. Despite this, no molecular or biological markers for selective transmission could be identified. A maternal neutralizing antibody response with broad specificity may protect the child from HIV-1 infection.

Synovial cells responding to a 65-kDa mycobacterial heat shock protein have a high proportion of a TcR gamma delta subtype uncommon in peripheral blood.

We have analysed the ability of T cells from synovial fluid mononuclear cells (SFMC) and from peripheral blood mononuclear cells (PBMC) of inflammatory arthritic diseases to proliferate in response to mycobacterial antigens (65‐kDa heal shock protein [hsp] of BCG. whole BCG) and to rat collagen type II. The SFMC demonstrated a significantly greater ability to respond to 65‐kDa hsp of BCG. and to whole BCG, compared with PBMC from the same patients, With collagen type II, only a small proportion of the patients showed a proliferative response, although with this antigen also SFMC responded better than PBMC. There was no difference between SFMC and PBMC in the response to control antigen (tetanus toxoid), phytohaemagglutinin (PHA), or interleukin 2 (IL‐2). A high proportion of cells in SFMC‐derived short‐term T‐cell lines were of TcRγδ type, often exceeding the number of TcRγδ type. There was a significantly higher proportion of TcRγδ cells in the SFMC lines compared with the PBMC lines, and a large part of the TcRγδ cells in she SFMC cultures was CD8+ The SFMC lines had a high proportion of δ‐ TCS‐1+ cells (Vδ1) among their TcRγδ cells, always exceeding the percentages of TiγA+ (Vγ9) and BB3+ (Vδ2) in the PBMC lines, the distribution of TcRγδ subtypes was markedly different, with a TiγA+/BB3+ population in the majority. These data argue for a different subpopulation distribution of TcRγδ cells in synovial fluid compared with peripheral blood of patients with inflammatory arthritic diseases.

Correlation between HIV sequence evolution, specific immune response and clinical outcome in vertically infected infants

Objective:To evaluate sequence evolution in relation to different rates of disease progression in infants infected with HIV-1. Design:Variability in the gp120 V3 region was analysed in HIV-1-infected children with different clinical courses, slow progression (n = 2) versus progressive disease (n = 3). Methods:Cloning and sequencing of virus-derived DNA from uncultured peripheral blood mononuclear cells was performed at two to three timepoints from birth and up to the fifth year of life. Sequence variability was estimated by calculating the genetic distance and the proportion and ratio of synonymous and non-synonymous nucleotide substitutions over time. Results:Genetic distances were significantly shorter in children with fast progression to disease, a predominance of synonymous nucleotide substitutions also being detected at later timepoints. Conversely, a preferential accumulation of non-synonymous nucleotide substitutions was apparent in children with slow disease progression. Furthermore, a positive correlation between a decreased ratio of synonymous/non-synonymous nucleotide substitutions and the ability of childrens sera to react with synthetic peptides representing the autologous virus sequence was determined. Conclusion:Data suggest that an antigenically more diverse virus population emerges in infected children with slower progression to disease as a result of a stronger immune pressure.

Mechanisms of escape from CD8+ T‐cell clones specific for the HER‐2/NEU proto‐oncogene expressed in ovarian carcinomas: Related and unrelated to decreased MHC class 1 expression

We have developed an in vitro model to study mechanisms by which ovarian tumor cells that over‐express the HER‐2/neu proto‐oncogene escape recognition by TCD8+. Nine tumor‐specific, HLA A2‐restricted TCD8+ clones were isolated from 2 ovarian tumor‐specific TCD8+ lines derived from tumor‐infiltrating or ‐associated lymphocytes. Of these, 2 clones recognized the previously defined HER‐2/neu epitope E75 (a.a. 369–377) and one recognized the C85 epitope (a.a. 971–979), whereas the specificity of the remaining 6 clones was unknown. Three different tumor escape variants (EVC8, EVC22 and EVC36) were produced by co‐culturing an ovarian tumor line over‐expressing HER‐2/neu with these autologous TCD8+ clones. Cell surface expression of HLA A2 was markedly decreased on all 3 escape variants, relative to the parental tumor line, while no significant decrease in their expression of the HER‐2/neu, ICAM‐1 or LFA‐3 molecules was found. There was a correlation between the level of tumor‐specific recognition and HLA A2 expression among the tumor clones isolated from 2 of the escape variants (EVC8 and EVC36). In contrast, high HLA A2‐expressing tumor clones isolated from the EVC22 variant, or EVC22 which had regained high HLA A2 expression through IFN‐γ treatment, were not recognized by the HER‐2/neu‐specific TCD8+ clone C‐22. No mutations were found in the cDNA or the genomic DNA derived from the PCR product corresponding to a 496 bp fragment including the region coding for the E75 epitope of the HER2/neu gene in the EVC22 variant. Collectively, this in vitro model underlines the importance of decreased expression of the HLA restriction element for escape from tumor‐specific TCD8+ but also demonstrates that additional mechanisms exist.

Restricted T cell receptor V-β and J-β usage in T cells from interleukin-2-cultured lymphocytes of ovarian and renal carcinomas

Tumour-infiltrating lymphocytes (TIL) are often observed in human tumours and their presence has been correlated with a better prognosis. It has been suggested that TIL are enriched for tumour-specific cytotoxic cells, and TIL activated and expanded in vitro by interleukin-2 (IL-2) are currently used in the therapy of human cancer. We have studied the T cell repertoire in IL-2-expanded TIL cells from patients with ovarian and renal carcinoma using T-cell-receptor-V-β-specific monoclonal antibodies and a polymerase-chain-reaction-based Southern blot technique for analysis of J-β usage. In TIL lines derived from three of nine patients with ovarian carcinomas and from two of eight patients with renal carcinomas, selective usage of the V-β6 or V-β5 T-cell receptor gene products was found. The majority of the cells were CD4+, with up to 40% of the T cells utilizing the same V-β gene. T-cell lines derived from peripheral blood lymphocytes from patients or healthy donors contained normal levels of V-β subsets. Only moderate levels of V-β6+ T cells were detected from freshly isolated TIL and the increase of this subpopulation appeared as a result of in vitro culture. The level of clonal restriction, as measured by the usage of J-β gene segments within the V-β5 or V-β6 families, was analysed using a recently developed technique based on the polymerase chain reaction. Evidence for restricted J-β usage was detected only in TIL expanded in vitro, while this was not the case in freshly isolated tumour-derived lymphocytes or T cell lines obtained from peripheral blood lymphocytes. The presence of a population with biased T cell receptor expression in cells derived from tumour tissue could be explained by their activation in vivo as a result of contact with tumour antigens and should be taken into consideration when discussing the therapeutic efficiency of IL-2-expanded TIL.

Clonal CD8+ and CD52- T cells are induced in responding B cell lymphoma patients treated with Campath-1H (anti-CD52).

Abstract:  Five patients with non‐Hodgkins lymphoma (NHL) and 4 patients with chronic lymphocytic leukaemia (CLL) were treated with the CDR‐grafted (rat × human) monoclonal antibody (mAb) Campath‐1H (anti‐CD52). Tumour regression was noted preferentially in peripheral blood and in the bone marrow but lymph nodes were less affected. Normal blood B and T cells were profoundly reduced in all patients whereas CD16+ NK cells and CD14+ monocytes decreased marginally. In all responding CLL patients CD52‐negative T but not B cells appeared during treatment and persisted for several months (4–19+) during unmaintained follow‐up. Clonal T cells defined as a predominance of a single T cell receptor (TCR) V gene usage, in one case verified by TCR CDR3 fragment analysis and nucleotide sequencing, emerged within the CD52–/CD8+ cell population during Campath‐1H therapy in 2 CLL patients, both achieving a long‐lasting remission. The increase in CD8+ T cell expansions (up to 23‐fold) during unmaintained remission and follow‐up suggest that the clonal CD8+ cells may represent regulatory T cells controlling the growth of the tumour B cell clone. Clonal T cells might thus be a target for an immune therapeutic intervention in B cell tumours.

Kinetics of the T-cell receptor CD4 and CD8 Vβ repertoire in HIV-1 vertically infected infants early treated with HAART

ObjectivesTo determine the kinetics and the relationship between the T-cell receptor Vβ (TCRBV) complementary determining region 3 length, the CD4 T-cell count and HIV viral load changes in HIV-1 infected infants treated early with highly active antiretroviral therapy (HAART) during 1 year of follow-up. DesignTwo HIV-1 vertically infected infants, two HIV-1 vertically exposed uninfected and two healthy controls were analysed by spectratyping. Evaluation of viral load, CD4 naive and memory cell counts and a proliferation test were also carried out. MethodsTwenty-six families and subfamilies of the TCR on CD4 and CD8 T cells were analyzed by spectratyping. Flow cytometric analysis on peripheral blood mononuclear cells for CD4CD45Ra, CD4CD45Ro, CD8CD38, proliferation tests and plasma viral load measurements were performed at baseline, 1, 6 and after 12 months of therapy. ResultsHAART induced a marked reduction of viral load in both HIV-1 infected infants and an increase to normal CD4 T-cell count in the symptomatic infant. At baseline the TCRBV family distribution in the majority of CD8 and a few of the CD4 T cells was highly perturbed, with several TCRBV families showing a monoclonal/oligoclonal distribution. During HAART a normalization of the TCR repertoire in both CD8 and CD4 subsets occurred. TCR repertoire normalization was associated with a good virological and immunological response. ConclusionThese results suggest that complete and early virus replication control as a result of early HAART leads to a marked reduction of T-cell oligoclonality and is an essential prerequisite to the development of a polyclonal immune response in HIV-1 infected infants.

Diverse T-Cell Receptor CDR3 Length Patterns in Human CD4+ and CD8+ T Lymphocytes from Newborns and Adults

T cells are essential in the initiation and maintenance of immune responses. Specific interaction between T cells and a presumptive antigen occurs through recognition of an MHC—peptide complex by the T‐cell receptor (TCR). The complementarity‐determining region (CDR) 3 of the TCR has direct contact with the peptide. Here we describe CDR3 length variability of six different TCRBV gene families of CD4+ and CD8+ umbilical cord (UC) and peripheral blood (PB) T cells. Amplified products spanning the TCR CDR3 regions from CD4+ PB, CD4+ UC and CD8+ UC blood T cells typically displayed Gaussian‐like distributions. In contrast, profound and frequent perturbations were recorded in CD8+ PB lymphocytes, with a non‐Gaussian pattern in more than half of the samples studied. A substantial portion of the perturbed CD8+ subsets were clonal or oligoclonal, as determined by CDR3‐length restriction, TCRBJ gene usage and nucleotide sequencing. This implies that the conditions for shaping and maintenance of the peripheral TCR repertoire are profoundly different for CD8+ and CD4+ T cells.

Detection of CD8 T-cell expansions with restricted T-cell receptor V gene usage in infants vertically infected by HIV-1.

Objective: To investigate the T‐cell receptor (TCR) repertoire usage in infants born to mothers infected with HIV‐1 in order to discern possible perturbations in TCR usage as a consequence of HIV‐1 infection. Design: Blood samples from five HIV‐1 ‐infected and six non‐infected children born to HIV‐1‐seropositive mothers were collected at two to three timepoints during the first and second year of life and the TCR variable gene usage was determined. Methods: Triple staining flow cytometry analysis using a panel of monoclonal antibodies (MAb) to TCR V&agr; and V&bgr; gene products and antibodies to CD4 and CD8 was performed. Results: Frequent large expansions of CD8+ lymphocyte subpopulations bearing distinct V&agr; and V&bgr; gene products was seen in HIV‐1‐infected children (four out of five) but was rarely detected in uninfected children. Conclusion: The study demonstrated the frequent occurrence of persistent and clonal expansions of CD8+ T cells bearing distinct V&agr;/V&bgr; gene products in some HIV‐1 vertically infected infants similar to those observed during primary infection in adults.

Role of immunity in maternal—infant HIV-1 transmission

Factors influencing human immunodeficiency virus type 1 (HIV‐1) mother‐to‐child transmission include both immunological and virological parameters: higher viral loads have been associated with clinical stage of HIV‐1‐infected individuals as well as higher risk of mother‐to‐child transmission. Furthermore, we have shown that transmitting mothers more frequently harbour HIV‐1 isolates with rapid/high syncytium‐inducing (SI) biological phenotype than non‐transmitting mothers do. Genetically homogeneous virus populations have been found in HIV‐1‐infected children at birth, in contrast to the heterogeneous virus populations often found in their infected mothers. This observation suggests that a few virus variants are transmitted or initially are replicating in the child. By comparing the HIV‐1 gp120 V3 region of sequentially obtained samples from infected children with samples obtained from their mothers at delivery we found, however, that multiple variants of HIV‐1 with different outgrowth kinetics can be transmitted. In addition, we have obtained results indicating an impaired ability of the immune response to adapt to the sequence evolution of HIV‐1 in transmitting mothers, as assessed by measuring serum reactivities to peptides representing selected yet closely related V3 sequences. By analysing the presence of antibodies in maternal serum at delivery, which neutralize autologous isolates as well as other primary virus isolates, we have indications that a protective immunity in HIV‐1 mother‐to‐child transmission might exist. Immunotherapy has been assessed in infected adult individuals by passive immunization with a variety of HIV‐1‐specific antibody products. Data from these studies indicated a differential response to therapy according to the stage of the disease. Active vaccine strategies, including envelope glycoproteins, pursued so far in seronegative adult subjects have shown limitations because broadly neutralizing antibodies, such as can be found in infected individuals, have not been evoked. Further investigations are therefore needed to give support for the potential use of either passive and/or active immunization for the prevention of HIV‐1 mother‐to‐child transmission.