HIV-1 infection in infants and the unidentified susceptibility of white blood cells for retroviral infection

Background: The immune system in the newborn is immature, as the lymphocytes have not yet encountered micro-organisms. This is reflected by the presence of non-activated naïve T-lymphocytes (CD45RA+ CD27+ CCR7+). Recently we have described that lymphocyte counts in uninfected children born to HIV-infected mothers initially increase after birth when infants are exposed e.g. to environmental micro-organisms. This process results in healthy adults in a maturation of the immune system with the occurrence of memory T lymphocytes (CD45RO+ CD27+ CCR7+/-). HIV-1 is a virus that attacks the immune system by infecting predominantly CD4+ T cells. The virus binds to CD4 and co-receptors on the surface membrane of CD4+ T cells to enter the cell. Under normal conditions, the obligatory co-receptor for early HIV-1 entry, i.e. chemokine receptor CCR5, is not expressed on naive CD4+ T cells present in the neonate. However, neonates or even a fetus with hardly if any memory CD4+ T cells can become infected with HIV-1 through maternal-to-child-transmission (MTCT). HIV-1 infection in neonates also progresses more rapidly than in adults with higher plasma viral loads (>10*6 copies per ml), and an increased risk on treatment failure of HAART. Thus, although low if any numbers of CCR5+ T cells are present, rapid infection and replication of HIV-1 does occur in newly infected neonates and infants. The aim of this project is to resolve this paradox. Hypothesis: HIV-1 infection occurs in CD4+ T cells that are cycling because they have recently been stimulated by cognate antigen or growth factors. Consequently, T cells stimulated through the T-cell antigen receptor (TCR) or homeostatic cytokine receptors attain characteristics of memory lymphocytes and rapidly become CCR5+ to serve as HIV targets. Alternatively, another set of white blood cells different from T cells is initially infected with HIV-1, resulting in rapid replication of the virus within these cells. Objectives: To elucidate the cellular components for HIV-1 infection. Methods and Design: First, to verify the reported lack of CCR5 on T cells at birth, we will compare an equal number of cord blood samples from HIV-exposed neonates and healthy non-exposed neonates for phenotyping of CD45RA/RO and chemokine receptor expression (e.g. CCR5) by multicolor flowcytometry. Follow-up samples at 1 month will be taken (in HIV-exposed neonates). Second, to define CCR5 upregulation upon short-term activation of naïve T cells from cord blood of healthy neonates by proliferative stimulation via TCR or by the growth factors IL-2, IL-7 and IL-15, we will determine surface expression and mRNA for chemokine receptors. Third, to determine productive HIV-1 infection rates in vitro of naïve T cells and other white blood cells in cord blood of healthy neonates, we will compare these infection rates with those of naïve and memory CD4+ T cells from healthy adult individuals, using highly purified cell fractions (MiniMACs colums, Miltenyi, Sanquin) and standard infection with CCR5-dependent HIV-1 (R5) strains. Fourth, analyze the inhibitory potential of CCR5 antagonists and monoclonal antibodies to prevent infection of highly purified naïve T cells (dose-response). Fifth, to assess the repertoire of co-receptors for HIV-1 infection on CD4+ T cell subsets from very young patients (and controls), i.e. CCR5, CCR2 and CXCR4 expression, we will perform extensive subset definition (by CD45RA/RO, CD27, CCR7, growth factor receptor proteins (CD25 [IL-2R-alfa], CD127 [IL-7R-beta], CD122 [IL-2R-beta] and CD132 [Vc]) and activation markers (CD38, HLA-DR) by multicolor flowcytometrie. Sixth, assessment of the genomic form of HIV-1 (integrated proviral or incomplete proviral DNA species, genomic RNA pre-integration complex) in purified naïve T cells of HIV-infected infants and compare with the form in memory T cell subsets and monocytes by PCR assays operational at Sanquin. Furthermore, viral characteristics of R5 HIV variants isolated from HIV-infected children will be defined (with respect to co-receptor usage, etc). Final goal: Generate solid data to identify the cells which are infected in pediatric HIV and the role of co-receptors and interleukins in the regulation of this infection. These data may clarify why high plasma viral loads occur in infected children and could also help to explain the increased risk on treatment failure of HAART in infants. Further insight in this mechanism may in the future contribute to the development of alternative treatment strategies for reduction of MTCT or treatment of HIV-infected (pediatric) patients.