Renu B. Lal

Use of inhibitors to evaluate coreceptor usage by simian and simian/human immunodeficiency viruses and human immunodeficiency virus type 2 in primary cells.

ABSTRACT We have used coreceptor-targeted inhibitors to investigate which coreceptors are used by human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency viruses (SIV), and human immunodeficiency virus type 2 (HIV-2) to enter peripheral blood mononuclear cells (PBMC). The inhibitors are TAK-779, which is specific for CCR5 and CCR2, aminooxypentane-RANTES, which blocks entry via CCR5 and CCR3, and AMD3100, which targets CXCR4. We found that for all the HIV-1 isolates and all but one of the HIV-2 isolates tested, the only relevant coreceptors were CCR5 and CXCR4. However, one HIV-2 isolate replicated in human PBMC even in the presence of TAK-779 and AMD3100, suggesting that it might use an undefined, alternative coreceptor that is expressed in the cells of some individuals. SIVmac239 and SIVmac251 (from macaques) were also able to use an alternative coreceptor to enter PBMC from some, but not all, human and macaque donors. The replication in human PBMC of SIVrcm(from a red-capped mangabey), a virus which uses CCR2 but not CCR5 for entry, was blocked by TAK-779, suggesting that CCR2 is indeed the paramount coreceptor for this virus in primary cells.

Activity of α- and θ-Defensins against Primary Isolates of HIV-1

θ-Defensins are lectin-like, cyclic octadecapeptides found in the leukocytes of nonhuman primates. They are also homologues of the more familiar α-defensins expressed by humans and certain other mammals. This study compares the ability of six θ-defensins (hominid retrocyclins 1–3 and rhesus θ-defensins 1–3) and four human α-defensins (human neutrophil peptides (HNPs) 1–4) to bind gp120 and CD4. In addition, we compared the ability of these θ-defensins and HNP-1 to protect J53-BL cells (an indicator cell line) from primary HIV-1 isolates that varied in subtype and coreceptor usage. The most potent θ-defensin, retrocyclin-2, bound with exceptionally high affinity to gp120 (KD, 9.4 nM) and CD4 (KD, 6.87 nM), and its effectiveness against subtype B isolates (IC50, 1.05 ± 0.28 μg/ml; 520 ± 139 nM) was approximately twice as great as that of HNP-1 on a molar basis. We also show, for the first time, that human α-defensins, HNPs 1–3, are lectins that bind with relatively high affinity to gp120 (KD range, 15.8–52.8 nM) and CD4 (KD range, 8.0–34.9 nM). Proteins found in human and FBS bound exogenous HNP-2 and retrocyclin-1, and competed with their ability to bind gp120. However, even the low concentrations of α-defensins found in normal human serum suffice to bind over half of the gp120 spikes on HIV-1 and a higher percentage of cell surface CD4 molecules. Although this report principally concerns the relationship between carbohydrate-binding and the antiviral properties of α- and θ-defensins, the lectin-like behavior of defensins may contribute to many other activities of these multifunctional peptides.

Protease sequences from HIV-1 group M subtypes A-H reveal distinct amino acid mutation patterns associated with protease resistance in protease inhibitor-naive individuals worldwide.

BackgroundAlthough numerous mutations that confer resistance to protease inhibitors (PRI) have been mapped for HIV-1 subtype B, little is known about such substitutions for the non-B viruses, which globally cause the most infections. ObjectivesTo determine the prevalence of PRI-associated mutations in PRI-naive individuals worldwide. DesignUsing the polymerase chain reaction, protease sequences were amplified from 301 individuals infected with HIV-1subtypes A (79), B (95), B’ (19), C (12), D (26), A/E (23), F (26), A/G (11), and H (3) and unclassifiable HIV-1 (7). Amplified DNA was directly sequenced and translated to amino acids to analyze PRI-associated major and accessory mutations. ResultsOf the 301 sequences, 85% contained at least one codon change giving substitution at 10, 20, 30, 36, 46, 63, 71, 77, or 82 associated with PRI resistance; the frequency of these substitutions was higher among non-B (91%) than B (75%) viruses (P < 0.0005). Of these, 25% carried dual and triple substitutions. Two major drug resistance-conferring mutations, either 20M or 30N, were identified in only three specimens, whereas drug resistance accessory mutations were found in 252 isolates. These mutations gave distinct prevalence patterns for subtype B, 63P (62%) > 77I (19%) > 10I/V/R (6%) = 36I (6%) = 71T/V (6%) > 20R (2%), and non-B strains, 36I (83%) > 63P (17%) > 10I/V/R (13%) > 20R(10%) > 77I (2%), which differed statistically at positions 20, 36, 63, 71, and 77. ConclusionsThe high prevalence of PRI-associated substitutions represent natural polymorphisms occurring in PRI-naive patients infected with HIV-1 strains of subtypes A−H. The significance of distinct mutation patterns identified for subtype B and non-B strains warrants further clinical evaluation. A global HIV-1 protease database is fundamental for the investigation of novel PRI.

Alternative Algorithms for Human Immunodeficiency Virus Infection Diagnosis Using Tests That Are Licensed in the United States

ABSTRACT Serodiagnosis of human immunodeficiency virus (HIV) infection in the United States has traditionally relied on a sequential two-test algorithm: an initial screen with an enzyme immunoassay (EIA) and reflex testing of EIA-reactive specimens with a more specific supplemental test such as Western blotting or immunofluorescence. The supplemental tests are tedious, subjective, and expensive. In addition, there have been major improvements in the performance and accuracy of the EIA tests as well as the introduction of rapid serologic tests (RT) and HIV nucleic acid amplification tests (NAAT). Related to these improvements is the possibility that alternative algorithms using combinations of currently approved HIV tests may function as well as if not better than the current algorithm, with more flexibility, improved accuracy, and lower cost. To this end, we evaluated the performance of 12 currently licensed tests and 1 in-house HIV test (6 EIA, 4 RT, and 3 NAAT) on panels of plasma samples from HIV-infected (n = 621 HIV type 1 [HIV-1] and 34 HIV-2) and uninfected (n = 513) people and of sequential specimens from people early in seroconversion (183 specimens from 15 patients). Test combinations were analyzed in two dual-test (sensitivity-optimized and specificity-optimized) algorithms and in a three-test (tie-breaking) algorithm, and performance was compared to the conventional algorithm. The results indicate that alternative algorithm strategies with currently licensed tests compare favorably with the conventional algorithm in detecting and confirming established HIV infection. Furthermore, there was a lower frequency of discordant or indeterminate results that require follow-up testing, and there was improved detection of early infection.

Concurrent infections and HIV pathogenesis.

The progression towards AIDS in HIV-1-infected individuals appears to be directly related to CD4 cell count decline and HIV-1 viral loads in the plasma and lymphoid tissues [1]. Though several factors account for the wide variations in HIV-1 disease progression, immune activation is probably the most important determinant for these differences. Activation of host immune cells (primarily CD4 cells, macrophages and dendritic cells) facilitates multiple steps of the viral life cycle and cellular factors that are directly or indirectly connected with it. These include up-regulation of the viral co-receptors (CCR5 and CXCR4), decreased âchemokine secretion [2], enhanced viral entry and integration as well as viral assembly and/or release [3]. Immune activation also leads to profound changes in the cytokine network, with increased secretion of tumor necrosis factor-alpha (TNF-á), interleukin (IL)2, IL-4, IL-6 and IL-10, and affects the cell cycle [4,5]. Immune activation is also associated with various degrees of immune dysfunction, hyporesponsiveness and apoptosis, all leading to enhanced progression of immune de®ciency and decreased survival [6±11]. Thus, since all concurrent infections, whether systemic or local, will lead to various degrees of immune activation in vivo, it is plausible that they may enhance HIV infection, increase HIV replication and viral load, and accelerate the progression of disease [12±14] (Fig. 1).

Plasmodium falciparum Antigen-Induced Human Immunodeficiency Virus Type 1 Replication Is Mediated through Induction of Tumor Necrosis Factor-α

Because malaria-stimulated cytokine production may have deleterious effects on human immunodeficiency virus type 1 (HIV-1) replication, the effects of Plasmodium falciparum antigens on HIV-1 replication were studied. Stimulation with malarial antigens significantly enhanced HIV-1 replication of HIV-1LAV and primary HIV-1 isolates (subtype A) in CD8-depleted peripheral blood mononuclear cells from naive donors. The malarial antigen-induced activation of HIV-1 was due to cellular activation as judged by the expression of cell activation markers and proliferative responses. While malarial antigen stimulation increased expression of tumor necrosis factor (TNF-alpha) and interleukin-6 (IL-6), only monoclonal antibodies (MAbs) to TNF-alpha inhibited malarial antigen-induced HIV-1 replication, whereas MAb to IL-6 had no effect. Malarial antigen increased HIV-1 replication by increasing viral mRNA expression and by activating long terminal repeat-directed viral transcription. These data suggest that P. falciparum infection can modulate HIV-1 pathogenesis by activating lymphocytes and stimulating viral replication through the production of cytokines.

Idiopathic CD4+ T-Lymphocytopenia -- An Analysis of Five Patients with Unexplained Opportunistic Infections

BACKGROUND Although patients with idiopathic CD4+ T-lymphocytopenia and serious opportunistic infections have been described previously, the clinical and immunologic features of this condition have not been well defined. METHODS We studied in detail five patients with idiopathic CD4+ T-lymphocytopenia. The studies included serologic testing, culture, and polymerase chain reaction for the human immunodeficiency virus (HIV) types 1 and 2, serologic testing for the human T-cell lymphotropic virus (HTLV) types I and II, lymphocyte phenotyping, immunoglobulin quantitation, and lymphocyte-transformation assays, as well as attempts to isolate a retroviral agent. The results were compared with those in HIV-infected persons matched for CD4+ T-cell counts and with those in normal controls. We also studied the spouses of patients and the blood donors for one patient. RESULTS In these five patients, there was no evidence of either HIV or HTLV infection. All the patients had both low percentages and low counts of CD4+ T cells, with relative increases in percentages, but not counts, of CD8+ cells. Numbers of B cells and natural killer cells were generally normal. As compared with HIV-infected persons, our patients had lower percentages and counts of CD8+ cells and more lymphopenia. CD4+ counts were relatively stable over time. Instead of the high immunoglobulin levels seen in HIV infection, these patients had normal or slightly low levels of immunoglobulins. The lymphocyte-transformation response to mitogens and antigens was depressed. Results in spouses and blood donors were normal. CONCLUSIONS Idiopathic CD4+ T-lymphocytopenia differs from HIV infection in its immunologic characteristics and in its apparent lack of progression over time. Nothing about the immunologic or viral-culture studies performed in these patients or about their family members or blood donors suggests that a transmissible agent causes this condition.

Adaptation to promiscuous usage of CC and CXC-chemokine coreceptors in vivo correlates with HIV-1 disease progression.

Objective:To study coreceptor usage of sequential primary HIV-1 isolates in a longitudinal follow-up cohort of HIV-1-infected men to understand its contribution to pathogenesis of HIV disease. Design:Viral coreceptor usage of sequential primary isolates from HIV-1-infected individuals was examined at various timepoints and data was compared with CD4 cell counts, rates of disease progression and β-chemokine production. Methods:Fifty-eight sequential primary isolates were obtained from four rapid progressors, six late progressors, and three long-term nonprogressors (LTNP) and their coreceptor usage was examined by infection of peripheral blood mononuclear cells (PBMC) from donors with wild-type or non-functional CC-chemokine receptor (CCR)-5, and by infection of GHOST4 cells expressing CD4 and various chemokine receptors [CCR-1–CCR-5, CXC-chemokine receptor (CXCR)-4, BOB/GPR15, BONZO/STRL33]. Production of RANTES and macrophage inflammatory protein (MIP)-1β was examined using unstimulated or phytohemagglutinin (PHA)-stimulated PBMC isolated from these individuals at multiple timepoints during infection. Results:A switch from single CCR-5 coreceptor usage to multiple coreceptor usage occurred in all four rapid progressors and three out of six late progressors. In addition to the commonly used coreceptors CXCR-4, CCR-5, and CCR-3, some of the viruses isolated from patients in the terminal stage of infection also used CCR-1, CCR-2b, CCR-4, and BOB as coreceptors. The emergence of viral variants capable of utilizing multiple coreceptors generally preceded CD4 cell decline to < 200 × 106/l and correlated with the onset of AIDS. In contrast, three LTNP maintained exclusive usage of CCR-5 over a period of 7–12 years post-infection. Endogenous production of RANTES and MIP-1β by PBMC from LTNP was not significantly different from rapid and late progressors. However, PHA-driven production of both chemokines was significantly higher in LTNP, suggesting that in vivo activating stimuli might curtail HIV replication by inducing these chemokines. Conclusions:Viral variants capable of utilizing a broad range of coreceptors correlated with HIV-1 disease progression. In contrast, LTNP maintain exclusive usage of CCR-5 and produce higher levels of β-chemokines. Thus, both viral and host determinants leading to the emergence of viral variants capable of using an expanded range of coreceptors may be likely determinants of disease progression.

Cervical and Prostate Primary Epithelial Cells Are Not Productively Infected but Sequester Human Immunodeficiency Virus Type 1

Primary prostate and cervical epithelial cells and epithelial cell lines were examined for human immunodeficiency virus type 1 (HIV-1) infection or transmission to peripheral blood mononuclear cells (PBMC). Neither cell-free nor cell-associated HIV-1 infected primary epithelial cells or cell lines. Pretreatment of HIV-1 to enhance CD4-independent entry did not augment infection. Cell surface expression was detected for galactosyl ceramide but not for CC-chemokine receptor 5, CXC-chemokine receptor 4, or CD4. The ability to transfer HIV-1 to resting or activated PBMC was tested by culturing with rinsed or trypsinized and replated HIV-1-exposed epithelial cells. Virus was not recovered from the rinsed or replated cocultures with resting PBMC; however, activated PBMC recovered HIV-1 from rinsed epithelial cells and rarely from replated epithelial cells. Although urogenital epithelial cells are not infected, these data suggest that they can transfer virus to activated immune cells and have implications for sexual transmission of HIV-1.

Male-to-female transmission of human T-cell lymphotropic virus types I and II : Association with viral load

SUMMARY Risk factors for male-to-female sexual transmission of human T-lymphotropic virus types I and II (HTLV-I/II) were investigated among HTLV-seropositive volunteer blood donors and their long-term (> or = 6 month) sex partners. Direction of transmission in concordantly seropositive pairs was assessed by analyzing risk factors for HTLV infection. Donors and their partners were also questioned regarding sexual behaviors during their relationships; HTLV antibody titers and viral load were determined for specimens from male partners. Among 31 couples in whom HTLV-infected men likely transmitted infection to their partners (11 HTLV-I and 20 HTLV-II) and 25 male-positive, female-negative couples (8 HTLV-I and 17 HTLV-II), HTLV transmitter men had been in their relationships longer (mean 225 months vs. 122 months) and had higher viral loads (geometric mean 257,549 vs. 2,945 copies/300,000 cells for HTLV-I; 5,541 vs. 118 copies/300,000 cells for HTLV-II) than non-transmitters (P = 0.018 and P = 0.001 for duration of relationship and viral load, respectively, logistic regression analysis). Transmitter men also tended to have higher antibody titers against various env and whole virus proteins than non-transmitters. The identification of high viral load and duration of relationship as risk factors provides a biologically plausible framework in which to assess risk of sexual transmission of the HTLVs.