Treatment with a rho kinase inhibitor improves survival from graft-versus-host disease in mice after MHC-haploidentical hematopoietic cell transplantation.

Acute graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT) and the main cause of nonrelapse mortality during the first 100 days post-transplant. Although GVHD can be prevented by extensive removal of mature donor T cells from the donor hematopoietic stem cell population, doing so eliminates any potential allogeneic graft-versus-tumor (GVT) effect also mediated by donor T cells and results in unacceptable rates of cancer relapse. One potential solution to this problem of separating GVHD development from a GVT response is to prevent T cell-mediated GVHD in the intestinal tract (IT) while preserving systemic antihost alloreactivity of donor T cells that target residual tumor cells expressing host alloantigens. We examined the ability of the anti-inflammatory rho kinase inhibitor, fasudil, given orally and intraperitoneally, to prevent GVHD in a C3H → B6C3F1 mouse model of MHC-haploidentical bone marrow transplantation. Fasudil-treated recipients of anti-thy-1 mAb + C' treated bone marrow (ATBM) cells plus T cells had a 73% 90-day survival compared with 25% among untreated ATBM + T cell recipients (P < .0001). Severe initial weight loss was similar in the 2 groups, but less diarrhea was observed among treated animals, and fasudil-treated survivors recovered more weight than untreated survivors. Skin inflammation occurred and resolved between weeks 2 and 8 with similar severity and kinetics in both treated and untreated surviving animals, indicating persistent alloreactivity. Day 10 post-transplantation splenocytes from fasudil-treated mice, containing mature donor T cells, and day 98 splenocytes, containing mature donor and de novo thymus-derived T cells, exhibited alloreactivity against host parental antigens, as assessed by in vitro IFN-γ production and rounds of allostimulated proliferation, respectively. These data support the idea that targeted treatment of the IT with rho kinase inhibitors can ameliorate lethal GVHD while preserving systemic alloreactivity. The results also suggest that similar mechanisms of IT-specific tolerance or resistance to GVHD operate in fasudil-treated and untreated long-term survivors of allogeneic ATBM + T cells.

Acquisition of CD4-dependence by CD4-independent SIV passaged in human peripheral blood mononuclear cells.

BACKGROUND: Chemokine receptors (CKRs), the primordial receptors for primate lentiviruses, are sufficient to mediate virus-cell fusion. Several different fusogenic CKRs and related receptors provide a broad potential host cell range, presumably advantageous for viral spread within a given infected individual, and across species. By contrast, the additional constraint of obligatory CD4 binding, just prior to CKR engagement, radically restricts potential host cells within an individual (or lymph node microenvironment), and might also limit xenotransmission, as CD4 sequences vary among primates. In spite of these potential drawbacks, CD4 dependent entry for SIV and HIV is the rule rather than the exception, and is generally thought to have evolved by selection for 1) stabilization of virus-cell surface interactions, and 2) conformational shielding of readily neutralized CKR binding epitopes. CD4 binding residues of SIV and HIV envelope are recessed, (relatively hidden from immune detection) and may exhibit a strong degree of automimicry, thus benefitting from self tolerance.Documented evolution, within individual macaques, of neutralization-resistant CD4-dependent SIV, derived from CD4-independent inocula, supports these ideas, but does not explain CD4's exclusive role as the penultimate receptor-even more striking, given the wide diversity of CKRs and other surface molecules that can serve as actual fusion receptors for SIV. We, therefore, explored the additional, non-exclusive, hypothesis that surface CD4 on leukocytes is a marker of a more favorable host cell environment, as compared to CD8, NK, or B cell surface markers. RESULTS: We demonstrate progressive in vitro evolution of two SIV strains to CD4-dependence (and CXCR4 tropism) in normal human PBMCs (hPBMCs). The two CD4-independent strains of SIV tested developed nearly complete CD4 dependence over several months of serial passage in hPBMCs, correlating with a limited number of non-synonymous env region mutations, some previously reported to be determinants of CD4-dependency. The initial ability of SIV stocks to grow to significant (albeit, relatively low) levels in CD4(-), CD14(-) cells was also lost with long term passage. Rapid emergence and subsequent prominence of G → A and A → G mutations within env regions associated with CD4 dependence was seen. CONCLUSIONS: Progressive acquisition of strict CD4 tropism, independent of immunoselection, supports the idea that surface CD4 identifies optimal host cells having intracellular environments most favorable to viral replication. The prominence of mutations involving G to A, or A to G, suggests that APOBEC 3 mediated infidelity may facilitate rapid switching of cell surface receptor usage within SIV swarms encountering fluctuating availability of optimal CD4+CKR+ targets. These observations of non-immune selection are compatible with, and may accelerate, simultaneous selection for previously described CD4-dependent neutralization resistance in vivo.

Anatomical loci of HIV-associated immune activation and association with viraemia.

BACKGROUND: Lymphocyte activation, associated with vaccination or infection, can be measured by positron emission tomography (PET). We investigated the ability of PET to detect and measure magnitude of lymph-node activation among asymptomatic HIV-1-infected individuals. METHODS: Initially we assessed PET response in eight HIV-1-uninfected individuals who had received licensed killed influenza vaccine. In an urban teaching hospital, we recruited 12 patients recently infected with HIV-1 (<18 months since seroconversion) and 11 chronic long-term HIV-1 patients who had stable viraemia by RT-PCR (non-progressors). After injection with fluorine-18-labelled fluorodeoxyglucose, patients underwent PET. We correlated summed PET signal from nodes with viral load by linear regression on log-transformed values. FINDINGS: Node activation was more localised after vaccination than after HIV-1 infection. In early and chronic HIV-1 disease, node activation was greater in cervical and axillary than in inguinal and iliac chains (p<0.0001), and summed PET signal correlated with viraemia across a 4 log range (r2=0.98, p<0.0001). Non-progressors had small numbers of persistently active nodes, most of which were surgically accessible. INTERPRETATION: The anatomical restriction we noted may reflect microenvironmental niche selection, and tight correlation of PET signal with viraemia suggests target-cell activation determines steady-state viral replication.

How do cell-free HIV virions avoid infecting dead-end host cells and cell fragments?

HIV faces the challenge of identifying and entering suitable host cells (i.e. activated and viable) among a wide array of receptor-positive but unsuitable targets. Lymph nodes contain resting cells, activated cells destined for apoptosis within 24 h, and cell fragments, all of which represent replicative dead ends. We postulate that 1) HIV virions have evolved the ability to probe the internal status of potential host cells from the external cell membrane by assessing the ability of cells to co-cap CD4 and chemokine receptors, and 2) the requirement for dual receptor binding in a concerted manner by three gp120 molecules is the molecular mechanism by which virions stochastically ensure high density co-capping of receptors. Cell-associated HIV accomplishes the same selective process by targeting cells capable of participating in immunological synapse formation.

Potentiation of EBV-induced B Cell transformation by CXCR4-tropic, but not CCR5-tropic, HIV gp120: implications for HIV-associated lymphomagenesis.

Abstract R5 and X4 HIV strains use CCR5 or CXCR4 chemokine receptors (CKRs), respectively, for entry. Preferential growth of X4 vs. R5 HIV in cell lines reflects constitutive expression of CXCR4, but not CCR5 (in contrast to dual expression on primary T cells), and CXCR4 is the predominant CKR found on most tumors. Non-Hodgkin's B cell lymphomas (NHL) are increased among HIV(+) patients, and interactions between HIV envelope and CKRs may contribute to lymphomagenesis. Despite strong evidence for a CXCR4-SDF-1 oncogenic axis, no in vitro evaluation of CXCR4-mediated normal lymphocyte transformation has been published. Exposure of normal B cells to EBV in the presence of X4 gp120 (but not R5 gp120) increased proliferation and BLCL outgrowth, comparable to anti-CD40 mAb costimulation. This suggests a role for X4 tropic viral envelope signaling via CXCR4 and/or CXCR7 in HIV-associated lymphomagenesis.

Risk associated HIV-1 cross-clade resistance of whole peripheral blood mononuclear cells from exposed uninfected individuals with wild-type CCR5.

Highly HIV exposed, persistently uninfected individuals (EUs) may hold clues to the generation of effective vaccine induced acquired immunity against HIV, and considerable effort has been devoted to detecting and characterizing HIV specific immune responses in EU cohorts. When searching for such clues, it is important to exclude individuals with genetically determined absence of receptors, as this protective mechanism could not be induced by HIV specific vaccines. Homozygosity for the DeltaC32 mutation of CCR5 prevents R5 HIV infection, independent of any virus-specific immune responses that may be acquired by exposure, while heterozygosity influences susceptibility to low level exposure. Reports on the in vitro susceptibility of EU cells compared to controls have been conflicting. Therefore, we studied 14 EUs with homozygous wild type CCR5, using a newly developed in vitro challenge assay (IVCA) to measure the magnitude and breadth of resistance to infection among EUs. CD8+ cells were relatively increased compared to controls, and were largely responsible for resistance to challenge, which depended on dose of virus inoculum, and extended across clades. Consistent with some EU cohort studies, resistance waned among individuals who reduced their high-risk behavior.

Modeling partially effective HIV vaccines in vitro.

Significant public health benefits could be realized with human immunodeficiency virus (HIV) vaccines that are incompletely effective. However, standard assays of experimental HIV vaccine immunogenicity may not correlate with antiviral effectiveness and cannot identify subtle effects. We developed an in vitro challenge assay (IVCA) that measures the net antiviral effect in whole peripheral blood mononuclear cells (PBMCs) to any titered HIV isolate. We then modeled partially effective postvaccination immune status 4 ways: use of PBMCs from highly exposed, uninfected individuals; depletion and partial reconstitution of autologous CD8+ cells from PBMCs from HIV-positive long-term nonprogressors; partial blocking of infection with chemokines; and variation in challenge virus dose. IVCA could detect as little as 3-fold differences in the challenge titer (30, 10, and 3 50% tissue-culture infective doses) or odds ratio of HIV infection. This robust and simple assay should be useful in determining which HIV vaccine candidates are suitable for field trials of efficacy.

HIV-2 subtype circulating in India (south).

The genetic types and subtypes of HIV vary from region to region. This study looked at the relative prevalence of HIV-2 subtypes in south India. Nucleotide sequencing of the HIV-2 envelope V3 region of strains from 11 individuals infected with HIV-2 and one individual who had dual infection with HIV-1 and HIV-2 was performed. Phylogenetic analysis showed that all individuals were infected with HIV-2 subtype A. These strains were from individuals who live in different regions of south India. In all, up to present, 17 strains inclusive of the authors' 12 have been sequenced, and subtype A of HIV-2 is seen in both monoinfections and dual infections with HIV-1 in India.

Safety and immunogenicity of a high-titered canarypox vaccine in combination with rgp120 in a diverse population of HIV-1-uninfected adults: AIDS Vaccine Evaluation Group Protocol 022A.

To test the safety and immunogenicity of a high-titered preparation of ALVAC-HIV vCP205 in both high-risk and low-risk persons and to evaluate variations in dosing schedule, we conducted a multicenter, randomized, double-blind trial of this vector in combination with recombinant subunit gp120 in 150 HIV-1-seronegative volunteers. The high-titered ALVAC vaccine was well tolerated; adverse events were minimal and not influenced by dosing. At day 728, the cumulative probability of a cytotoxic T-lymphocyte (CTL) response was 76% (95% confidence interval [CI]: 64%-89%) among volunteers receiving vaccine, and the net amount attributable to vaccination was 50% (CI: 16%; 74%). The net probability of a repeated positive CTL response by day 728 was 50% (CI: 21%; 64%). There was a significant difference in CTL response at day 182 between volunteers who had received four doses versus three doses of vCP205 (42% vs. 24%, p =.052). The CTL response was similar in high-risk volunteers and vaccinia-naive volunteers compared with vaccinia-immune volunteers. Neutralizing antibody responses were detected in 95% of vaccinees at day 287, with higher geometric mean titers in recipients of sequential versus simultaneous dosing of the two vaccines and in vaccinia-naive volunteers. This high-titered preparation of ALVAC-HIV vCP205 in combination with gp120 was safe and immunogenic in a diverse group of HIV-1-seronegative volunteers.