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Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel θ-defensin retrocyclin-101 (RC-101) against flavivirus infection and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the nonstructural protein NS2B-NS3 serine protease might serve as a potential viral target. Furthermore, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III (DIII), which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection. IMPORTANCE Retrocyclin is an artificially humanized circular θ-defensin peptide, containing 18 residues, previously reported to possess broad antimicrobial activity. In this study, we found that retrocyclin-101 inhibited flavivirus (ZIKV and JEV) infections. Retrocyclin-101 inhibited NS2B-NS3 serine protease activity, suggesting that the catalytic triad of the protease is the target. Moreover, retrocyclin-101 bound to the DE loop of the E protein of flavivirus, which prevented its entry.

Inflammation is an integral component of autoimmune arthritis. The balance of pathogenic T helper 17 (Th17) and protective T regulatory (Treg) cells can influence disease severity, and its resetting offers an attractive approach to control autoimmunity. We determined the frequency of Th17 and Treg in the joints of rats with adjuvant arthritis (AA), a model of rheumatoid arthritis (RA). We also investigated the impact of Celastrol, a bioactive compound from the traditional Chinese medicine Celastrus that can suppress AA, on Th17/Treg balance in the joints. Celastrol treatment reduced Th17 cells but increased Treg in the joints, and it inhibited Th17 differentiation but promoted Treg differentiation in vitro by blocking the activation of pSTAT3. Furthermore, Celastrol limited the production of Th17-differentiating cytokines and chemokines (CCL3, CCL5). Thus, Celastrol suppressed arthritis in part by altering Th17/Treg ratio in inflamed joints, and it should be tested as a potential adjunct/alternative for RA therapy.

When attacked by HIV, the immune system counteracts infection with elicitation of HIV-specific antibodies and cytotoxic T lymphocytes. In most cases however, these defenses are unable to resolve HIV infection, which progresses, if left untreated, ravaging the immune system and leading to AIDS and, eventually, to death. Nonetheless, there are additional components of the immune system, from both the innate and the adaptive components, that are associated with improved clinical status and, in some cases, even with protection from infection. Two distinct families of such factors have been studied in depth: chemokines and beta-defensins. CCR5 chemokines, which are involved in adaptive immunity, are molecules produced by lymphocytes, and thus are likely to play a role in controlling HIV systemically. beta-defensins are instead produced by epithelial cells, and thus are important in controlling infection at mucosal sites. Both of these families of molecules, therefore, are involved in crucial battlegrounds for fighting HIV infection. Here, we review the evidence that argues for their importance in AIDS pathogenesis and in preventive and therapeutic approaches to combat HIV infection.

Oral transmission of human immunodeficiency virus type 1 (HIV-1) is poorly understood, partly because data on this type of transmission are scarce and inherently problematic to analyze and interpret. Despite recent suggestions that the risk of becoming infected through the oral route may be higher than previously estimated, oral transmission of HIV is 8 to 10 times less likely to occur than vaginal or rectal transmission in humans (41, 50, 61). This is in contrast with studies performed in infant macaques, which are readily infected orally with simian immunodeficiency virus (3, 42). Therefore, it is possible that the oral mucosa in infants has a critical defect in immunity that renders them more susceptible to viral infection. A better understanding of the protection mechanism in the oral environment will likely provide information useful for preventing mother-to-child transmission of HIV-1. Although the scarcity of data renders it difficult to envision a model for protection in the oral mucosa, there are a number of possible explanations for the relatively low rate of oral transmission of HIV (41, 50, 61). One possibility is that inhibitory factors are responsible for this phenomenon. Interestingly, saliva has been shown to inhibit HIV-1 replication, and several salivary proteins, including anti-HIV antibodies, mucins, thrombospondin, lysozyme, lactoferrin, lactoperoxidase, cystatins, complement, statherin, and secretory leukocyte protease inhibitor, have been characterized as HIV inhibitors (41, 50, 61). Other inhibitory proteins might also reside in the mucosa. β-Defensins, a subfamily of homologous antimicrobial peptides constituting an important component of innate immunity found predominantly in vertebrates, are among the proteins expressed at the highest levels in the oral mucosa (17, 20, 29, 33, 62). Human defensins are cationic and Cys-rich proteins with molecular weights ranging from 3 to 5 kDa. Based on sequence homology and the connectivity of six conserved cysteine residues, human defensins are classified into α and β families. Human α-defensins were first discovered as natural peptide antibiotics (HNP1 to HNP4) stored in the azurophilic granules of neutrophils and released to combat ingested foreign microbes during phagocytosis (16, 48, 56). Other α-defensins, secreted in response to bacterial stimulation, have also been found in intestinal Paneth cells (2, 26, 27). In contrast, human β-defensins (hBDs) are found predominantly in various epithelial cells and tissues (4, 46, 64). hBD1, originally isolated from human blood filtrate (4), is constitutively expressed in the urogenital and airway tracts, suggesting a role in protecting mucosae from microbial infection (64). hBD2, first isolated from lesions of inflamed skin, is transcriptionally up-regulated by inflammatory stimuli such as cytokines and microorganisms (21). hBD3 was isolated originally from inflamed human skin, is inducibly expressed in various epithelia (18, 22, 25, 47), and possesses a broad spectrum of potent bactericidal activities in a salt-insensitive manner against both gram-positive and gram-negative bacteria (18, 22, 25, 46). Genomics studies have identified several new members of the β-defensin family (28, 40, 53). Recently, a circular form of antimicrobial peptides, called θ-defensins, has been characterized in macaques, while in humans all the genes encoding these peptides contain stop codons and are therefore likely to constitute expressed pseudogenes (12). Despite their diversity, defensins kill a broad range of microorganisms including bacteria, fungi, and certain enveloped viruses, presumably through membrane disruption, and constitute an important component in innate immunity. Thus, they could also affect HIV-1 infection by interfering with its envelope. All classes of defensins reportedly can suppress HIV replication (9, 30, 32, 39, 58, 63). Nakashima et al. described α-defensins as antiviral in 1993 (32), and recent work had suggested that they might play a role in controlling HIV in some subjects (63). θ-Defensins, present only in nonhuman primates, might be part of the cross-species barrier to HIV. Recently, the anti-HIV activity of β-defensins has also been reported (39). However, for two reasons, it is difficult to extrapolate the antiviral activity of α- and θ-defensins to resistance against oral HIV transmission. First, θ-defensins are not expressed in humans (12). Second, α-defensins are not prominently expressed in the oral mucosa (13, 15). In contrast, β-defensins are highly expressed in the oral epithelium (13, 15, 43), with measured local concentration as high as 100 μg/ml in a 100-μm-thick layer (49). Therefore, β-defensins are candidates as components of innate resistance to oral HIV infection. In this report, we show that hBD2 and hBD3 inhibit HIV-1Bal, an R5 isolate, and IIIB, an X4 isolate; hBD2 exerted its antiviral activity without affecting cellular proliferation. Further, our data indicate that inhibition of HIV occurs at an early stage. Surprisingly, our data ruled out a mechanism involving inhibition of membrane fusion, including downregulation of HIV receptors. Instead, our data are consistent with a dual mechanism of inhibition. One component of the HIV-suppressive activity of hBD2 is due to a direct inactivation of virions, while a second component is observed postentry. Therefore, hBD2 inhibits HIV replication through a mechanism, possibly related to that described for α-defensins (9, 9a), which is not merely based on membrane disruption. These results indicate that the study of the antiviral activity of hBD2 and hBD3 could be useful in providing new tools for HIV prevention (for example, as topical microbicides), in therapy (either as such or as a basis for developing new drugs), and as correlates of immunity in the oral cavity.

We had previously reported the cloning of the complete genome of an isolate of hepatitis C virus (HCV), HCV-S1, of genotype 1b. We have constructed a full-length complementary DNA (cDNA) clone of HCV-S1 using nine overlapping cDNA clones that encompassed its entire genome. HCV core, E1, E2, NS-3, -4B, -5A, and -5B proteins were detected in 293T cells by immunoblot analyses when expression of the full-length HCV-S1 was driven under a CMV promoter. Expression of full-length HCV-S1 led to induction of the CC chemokines RANTES and MCP-1 at both the mRNA and the protein levels in HeLa, Huh7, and HepG2 cells. Reporter gene assays showed that a minimal MCP-1 promoter construct containing 128 nucleotides upstream of its translational start site was sufficient for optimal HCV-mediated activation. HCV induced AP-1 binding activities to this region, as determined from electrophoretic mobility shift assays and supershifts with anti-AP-1 antibodies. Transfection of full-length HCV-S1 up-regulated both AP-1 binding activities as well as c-jun transcripts. A minimal promoter construct containing 181 nucleotides upstream of the RANTES translational start site was sufficient for maximal HCV-mediated induction. Gel mobility shift and supershift assays showed that HCV induced NF-κB and other unknown binding activities to the A/B-site within this region. In HeLa cells, HCV core and NS5A could separately augment promoter activities of both MCP-1 and RANTES. In Huh7 cells, only NS5A produced a similar effect, while rather surprisingly, HCV core induced a dramatic reduction in promoter activities of these two genes. This study provides the first direct evidence for the induction of CC chemokines in HCV infection and draws attention to their roles in affecting the progress and outcome of HCV-associated liver diseases.

Chemokines that bind to human immunodeficiency virus (HIV) co-receptors are potent and selective inhibitors of HIV infection. Therefore, ever since our discovery of this activity, we have proposed their role in controlling HIV infection as a third arm of the immune response, i.e. in concert with humoral and cellular responses. Research carried out in our laboratory, and performed independently by other groups, has recently strengthened this concept. Here, we critically analyze the evidence indicating the positive contribution of chemokines to HIV infection, their involvement with cognate and innate immunity, and the potential for their use in combating HIV infection.

The beta-chemokines RANTES, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta suppress infection by macrophage-tropic strains of HIV and simian immunodeficiency virus (SIV) by binding and down-regulating the viral coreceptor, CCR5. Accordingly, we have examined whether higher levels of CCR5 ligands are associated with a more favorable clinical status in AIDS. A cross-sectional study of 100 subjects enrolled in the Multicenter AIDS Cohort Study at the Baltimore site was conducted to measure chemokine production and lymphocyte proliferation by peripheral blood mononuclear cells (PBMC). Statistical analyses of the data revealed that the production of HIV-suppressive beta-chemokines by HIV antigen-stimulated PBMC was significantly higher in HIV-positive subjects without AIDS compared with subjects with clinical AIDS. Increased chemokine production was also correlated with higher proliferative responses to HIV antigens. Both parameters were significantly lower in the AIDS versus non-AIDS group. Notably, significantly higher levels of MIP-1alpha were also observed with unstimulated PBMC from seronegative subjects at risk for HIV infection released as compared with seropositive and non-Multicenter AIDS Cohort Study seronegative subjects. The association of chemokine production with antigen-induced proliferative responses, more favorable clinical status in HIV infection, as well as with an uninfected status in subjects at risk for infection suggests a positive role for these molecules in controlling the natural course of HIV infection.

Several chemotactic cytokines, or chemokines. inhibit HIV replication by blocking or down regulating chemokine receptors that serve as entry cofactors for the virus. Although the role of chemokine receptors in HIV pathogenesis has been the subject of intense interest, chemokines are comparatively less seriously considered as potential correlates of protection from HIV infection and disease progression. However, a critical analysis of newly available data reveals substantial evidence to support a beneficial role for chemokines in HIV infection and disease. In this review we summarize the results of such studies and their promising implications for HIV infection.

CD8+T lymphocytes from individuals infected with human immunodeficiency virus–type 1 (HIV-1) secrete a soluble activity that suppresses infection by HIV-1. A protein associated with this activity was purified from the culture supernatant of an immortalized CD8+T cell clone and identified as the β-chemokine macrophage-derived chemokine (MDC). MDC suppressed infection of CD8+cell–depleted peripheral blood mononuclear cells by primary non–syncytium-inducing and syncytium-inducing isolates of HIV-1 and the T cell line–adapted isolate HIV-1IIIB. MDC was expressed in activated, but not resting, peripheral blood mononuclear cells and binds a receptor on activated primary T cells. These observations indicate that β-chemokines are responsible for a major proportion of HIV-1–specific suppressor activity produced by primary T cells.

Accelerated bone loss leading to osteopenia, osteoporosis, and bone fracture is a major health problem that is increasingly common in human immunodeficiency virus (HIV) infected patients. The underlying pathogenesis is unclear but occurs in both treatment naïve and individuals receiving antiretroviral therapies. We developed an HIV-1 transgenic rat that exhibits many key features of HIV disease including HIV-1 induced changes in bone mineral density (BMD). A key determinant in the rate of bone loss is the differentiation of osteoclasts, the cells responsible for bone resorption. We found HIV-1 transgenic osteoclast precursors (OCP) express higher levels of suppressor of cytokine signaling-1 (SOCS-1) and TNF receptor associated factor 6 (TRAF6) and are resistant to interferon-gamma (IFN-γ) mediated suppression of osteoclast differentiation. Our data suggest that dysregulated SOCS-1 expression by HIV-1 transgenic OCP promotes osteoclastogenesis leading to the accelerated bone loss observed in this animal model. We propose that elevated SOCS-1 expression in OCP antagonizes the inhibitory effects of IFN-γ and enhances receptor activator of NF-kB ligand (RANKL) signaling which drives osteoclast differentiation and activation. Understanding the molecular mechanisms of HIV-associated BMD changes has the potential to detect and treat bone metabolism disturbances early and improve the quality of life in patients.

Background Human Immunodeficiency Virus Type I (HIV-1) infection is associated with a high incidence of B-cell lymphomas. The role of HIV in these lymphomas is unclear and currently there are no valid in vivo models for better understanding HIV-related lymphomagenesis. Transgenic (Tg) 26 mice have a 7.4-kb pNL4-3 HIV-1 provirus lacking a 3.1-kb sequence encompassing parts of the gag-pol region. Approximately 15% of these HIV Tg mice spontaneously develop lymphoma with hallmark pre-diagnostic markers including skin lesions, diffuse lymphadenopathy and an increase in pro-inflammatory serum cytokines. Here we describe the phenotypic and molecular characteristics of the B cell leukemia/lymphoma in the Tg mice. Results The transformed B cell population consists of CD19+pre-BCR+CD127+CD43+CD93+ precursor B cells. The tumor cells are clonal and characterized by an increased expression of several cellular oncogenes. Expression of B cell-stimulatory cytokines IL-1β, IL-6, IL-10, IL-12p40, IL-13 and TNFα and HIV proteins p17, gp120 and nef were elevated in the Tg mice with lymphoma. Conclusions Increased expression of HIV proteins and the B-cell stimulatory factors is consistent with the interpretation that one or more of these factors play a role in lymphoma development. The lymphomas share many similarities with those occurring in HIV/AIDS+ patients and may provide a valuable model for understanding AIDS-related lymphomagenesis and elucidating the role played by HIV-1.

Evidence suggests that CD8+ T lymphocytes are involved in the control of human immunodeficiency virus (HIV) infection in vivo, either by cytolytic mechanisms or by the release of HIV-suppressive factors (HIV-SF). The chemokines RANTES, MIP-1 alpha, and MIP-1 beta were identified as the major HIV-SF produced by CD8+ T cells. Two active proteins purified from the culture supernatant of an immortalized CD8+ T cell clone revealed sequence identity with human RANTES and MIP-1 alpha. RANTES, MIP-1 alpha, and MIP-1 beta were released by both immortalized and primary CD8+ T cells. HIV-SF activity produced by these cells was completely blocked by a combination of neutralizing antibodies against RANTES, MIP-1 alpha, and MIP-1 beta. Recombinant human RANTES, MIP-1 alpha, and MIP-1 beta induced a dose-dependent inhibition of different strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV). These data may have relevance for the prevention and therapy of AIDS.

s-defensins are antimicrobial peptides secreted by epithelial cells that can bind to cellular receptors. CCR2 and CCR6 are the two cellular receptors known to bind human s-defensin (hBD) 2 and -3. Both of these receptors are of crucial relevance in HIV infection. CCR6 is expressed, often in concert with CCR5, on cells that are highly susceptible to HIV infection: memory T cells, Th17 cells, α4s7+ cells, and defects in CD4+CCR6+ cells have been associated with faster AIDS progression. CCR2 is expressed on monocytes and macrophages, cells that are reservoirs of HIV infection and that are known to mediate central nervous system damage. Our studies show that hBD2, hBD3, and CCR6 ligand MIP-3α/CCL20 inhibit HIV infection via CCR6 by increasing expression of the antiviral protein APOBEC3G. This increase is due to a transcriptional mechanism mediated by intracellular signaling. hBD2 also inhibits HIV replication in macrophages that express CCR2. Our findings suggest novel therapeutic and preventive approaches that exploit CCR6 and CCR2-mediated intracellular signaling to inhibit HIV infection in highly susceptible cells.

T-cell-derived soluble factors that inhibit both X4 and R5 HIV are recognized as important in controlling HIV. Whereas three β chemokines, regulated-on-activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β, account for the suppression of R5 HIV by blockade of HIV entry, the major components responsible for the inhibition of X4 HIV strains have not been identified previously. We identify these factors primarily as a mixture of three β chemokines [macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), and I-309] and two RNases (angiogenin and RNase 4) of lesser potency and show that in a clade B population, some correlate with clinical status and are produced by both CD4 + and CD8 + T cells (chemokines, angiogenin) or only by CD8 + T cells (RNase 4). The antiviral mechanisms of these HIV X4-suppressive factors differ from those of the previously described HIV R5-suppressive β chemokines.

The Th17/interleukin (IL)-17 axis controls inflammation and might be important in the pathogenesis of experimental autoimmune myocarditis (EAM) and other autoimmune diseases. However, the mechanism underlying the increased Th17 cell response in coxsackievirus-induced myocarditis remains unclear. This study aimed to elucidate the regulatory mechanisms affected by blocking IL-17A responses in acute virus-induced myocarditis (AVMC) mice. The results showed that IL-17A and COX-2 proteins were significantly increased in the cardiac tissue of acute myocarditis, as were Th17 cells in the spleen. Using anti-mouse IL-17Ab to block IL-17A on day 7 of the viral myocarditis led to decreased expressions of cardiac tumor-necrosis factor alpha, IL-17A and transforming growth factor beta in AVMC mice compared to isotype control mice. COX-2 and prostaglandin E2 proteins were dramatically elevated, followed by marked reductions in CVB3 replication and myocardial injury. These results hint that the Th17/IL-17 axis is intimately associated with viral replication in acute myocarditis via induction of COX-2 and prostaglandin E2.

Neurotrophins control cell survival. Therefore, we examined whether HIV-1 reduces neurotrophin levels. Serum of HIV-positive individuals exhibited lower concentrations of brain-derived neurotrophic factor (BDNF), but not of other neurotrophins, than HIV-negative individuals. In addition, R5 and X4 strains of HIV-1 decreased BDNF expression in T cells. Our results support the hypothesis that reduced levels of BDNF may be a risk factor for T-cell apoptosis and for neurological complications associated with HIV-1 infection.

Circulating plasmacytoid dendritic cells (pDC) decline during HIV-1 infection, but at the same time they express markedly higher levels of interferon alpha (IFNalpha), which is associated with HIV-1 disease progression. Here we show an accumulation of pDC in lymph nodes (LN) of treatment-naïve HIV-1 patients. This phenomenon was associated with elevated expression of the LN homing marker, CCR7, on pDC in peripheral blood of HIV-1 patients, which conferred increased migratory capacity in response to CCR7 ligands in ex vivo functional assays. LN-homed pDC of HIV-1 patients presented higher CD40 and lower BDCA2 levels, but unchanged CD83 and CD86 expression. In addition, these cells expressed markedly higher amounts of IFNalpha compared to uninfected individuals, and were undergoing faster rates of cell death. These results demonstrate for the first time that in asymptomatic, untreated HIV-1 patients circulating pDC up-regulate CCR7 expression, accumulate in lymph nodes, and express high amounts of IFNalpha before undergoing cell death. Since IFNalpha inhibits cell proliferation and modulates immune responses, chronically high levels of this cytokine in LN of HIV-1 patients may impair differentiation and immune function of bystander CD4(+) T cells, thus playing into the mechanisms of AIDS immunopathogenesis.

The maladaptive Type C coping style has been linked to disease progression in HIV and other immunologically mediated disorders. We hypothesized that strong Type C coping, higher levels of alexithymia, and greater cardiovascular (particularly heart rate) responses to, and prolonged recovery from stress would be associated with poorer functioning of immune parameters previously linked to HIV pathogenesis and progression: (1) antigen-stimulated production of the beta (beta)-chemokines MIP-1 alpha and MIP-1 beta, which bind to the HIV co-receptor CCR5 and block HIV entry into CD4(+) lymphocytes; and (2) antigen-stimulated production of the proinflammatory cytokine interleukin-6 (IL-6), which synergizes immune activation associated with HIV replication. We examined relations among psychological, cardiovascular, and immune variables in a baseline sample of 200 HIV-infected, predominantly African American outpatients attending an HIV primary care clinic in inner-city Baltimore. In regression analyses adjusted for CD4(+) count and age, strong Type C coping was associated with significantly higher IL-6 production, as predicted. The theoretically related construct of alexithymia was correlated with significantly lower stimulated production of HIV-inhibiting MIP-1 alpha. Independent of alexithymia, greater heart rate reactivity, and poorer heart rate recovery in response to experimental stressors were also significantly associated with lower production of MIP-1 alpha, adjusted for cardiovascular medications, methadone use, CD4(+) count, and age. These findings support our primary set of hypotheses that maladaptive Type C coping, alexithymia, and heart rate reactivity/recovery are associated with disturbances in two key immune parameters implicated in HIV pathogenesis. Our secondary hypothesis, that dysregulated heart rate reactivity may mediate the connections between Type C coping and/or alexithymia and IL-6/ MIP-1 alpha was not confirmed. The finding that Type C coping, alexithymia, and heart rate reactivity/recovery are associated independently and differentially with specific aspects of relevant immune functioning may reflect distinct biobehavioral pathways that contribute to HIV progression.

Results The CD4+ T cells produced IFN-y and MIP-1a in response to gp120. Also detected were RANTES and MIP-1a, but not MDC (CCL22). The b-chemokines displayed potent inhibitory activities against R5-tropic HIV-1. After the cells were exposed to BX08 (an R5 subtype B HIV-1 primary isolate), the capacity of these cells to produce IFN-y and MIP-1a in response to gp120 was initially comparable with that of the unexposed control. But after 3–4 days of infection, the numbers of cells capable of producing IFNy and MIP-1a decreased significantly, while the percentage of infected cells reached >80%. Conclusion HIV-specific CD4+ T cells are susceptible to HIV. HIV reduces the capacity of the cells to produce IFN-y and bchemokines. On-going efforts to enhance HIV-specific CD4+ T cell resistance against the virus will be presented. from 2006 International Meeting of The Institute of Human Virology Baltimore, USA. 17–21 November, 2006

Background We have shown that the Type C style of coping with stress (diminished ability to recognize and express stress/ distress/emotions) is related to HIV progression. We recently found in a study of 50 HIV+ patients in Baltimore, that stronger Type C coping is associated with decreased production of beta-chemokines that bind to the HIV coreceptor CCR5. Under the aegis of NIH, we have initiated a longitudinal study with a final N = 200 to evaluate the core hypothesis that lower production of the beta-chemokines MIP-1 α/β mediates the relationship between Type C coping and HIV progression.

Chemokines are key players in the elicitation of immune response, by selectively attracting subpopulations of immune cells to the site of antigen presentation. Therefore, they are natural candidates for modulating immune responses to antigens qualitatively and quantitatively. We have selected chemokines associated with different arms of the immune response, i.e. RANTES/CCL5, B-lymphocyte chemoattractant/CXCL13, and monocyte chemoattractant protein-1/CCL2, and co-injected DNA expression constructs encoding these chemokines with constructs encoding two HIV antigens, gp120 and gp160, in mice. We subsequently measured markers of both cellular and humoral immune responses, and found that these chemokines qualitatively influenced the outcome of immune responses to both antigens, essentially according to their predicted association to Th profiles. These results are relevant towards the engineering of novel vaccine and immune-based therapies, and point to chemokines as candidate adjuvant and immunomodulatory molecules.

CCR5 ligands RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta are potent and specific inhibitors of strains of human immunodeficiency virus (HIV) that use CCR5 as a receptor, which are the strains most involved in primary infection. Recently, we observed that release of CCR5 ligands is a consistent and reproducible parameter of response to antigen activation in studies using PBMC. In this study, we show that CCR5 ligands are released upon antigen [Fragment C of tetanus toxin (TTC)] stimulation in 81% (n = 16) of subjects tested, as detected by a standard ELISA in tissue culture supernatants of antigen-activated cells. In contrast, ELISA for other cytokines from the same supernatants revealed that IFN-gamma release could be detected only in 31% of subjects, IL-2 could be detected only in 12% of the subjects and IL-4 was not detectable in any of the subjects tested. Similarly, proliferative responses to TTC, as measured by a standard tritiated thymidine incorporation assay, were detectable in only 56% of the subjects. Similar observations have been reported in flow cytometric studies, and resonate with previous findings emphasizing the role of CCR5 in T cell responses. In addition, the levels of CCR5 ligands in supernatants from antigen-activated cells were sufficient to inhibit infection of R5 HIV. Thus, CCR5 ligands might play a role in controlling HIV in vivo. Taken together, these observations suggest that CCR5 ligands, and in particular MIP-1alpha and MIP-1beta, released in the course of memory responses may play a role in protecting CD4(+) memory T cells from infection.

To test the hypothesis that β-chemokine levels may be relevant to the control of HIVin vivo, we compared RANTES, MIP-1α, and MIP-1β production from purified CD8+T cells from 81 HIV-infected subjects and from 28 uninfected donors. Asymptomatic HIV+subjects produced significantly higher levels of MIP-1α and MIP-1β, but not RANTES, than uninfected donors or patients that progressed to AIDS. In contrast, β chemokines in plasma were either nondetectable or showed no correlation with clinical status. The high β-chemokine-mediated anti-HIV activity was against the macrophage tropic isolate HIV-1BAL, with no demonstrable effect on the replication of the T-cell tropic HIV-1IIIB. These findings suggest that constitutive β-chemokine production may play an important role in the outcome of HIV-1 infection.

The recently cloned human chemoattractant receptor-like (CMKRL)1, which is expressedin vivoin CD4-positive immune cells, has structural homology with the two chemokine receptors C-C chemokine receptor (CCR)5 and C-X-C chemokine receptor (CXCR)4, which serve as the major coreceptors necessary for fusion of the HIV-1 envelope with target cells. In view of the structural similarity, CMKRL1 was tested for its possible function as another HIV-1 coreceptor after stable expression in murine fibroblasts bearing the human CD4 receptor. The cells were infected with 10 primary clinical isolates of HIV-1, and entry was monitored by semiquantitative PCR of viral DNA. The efficiency of the entry was compared with the entry taking place in CD4-positive cells expressing either CCR5 or CXCR4. Seven of the isolates used CMKRL1 for viral entry; they were mainly of the syncytium-inducing phenotype and also used CXCR4. Entry efficiency was higher with CMKRL1 than with CXCR4 for more than half of these isolates. Three of the ten isolates did not use CMKRL1; instead, entry was mediated by both CCR5 and CXCR4. The experiments thus indicate that CMKRL1 functions as a coreceptor for the entry of HIV-1 into CD4-positive cells. In the course of this study, leukotriene B4was shown to be the natural ligand for this receptor (now designated BLTR), which therefore represents a novel type of HIV-1 coreceptor along with the previously identified chemokine receptors. BLTR belongs to the same general chemoattractant receptor family as the chemokine receptors but is structurally more distant from them than are any of the previously described HIV-1 coreceptors.

The C-C chemokines RANTES, MIP-1alpha, and MIP-1beta have been characterized as constituents of an HIV- and SIV-suppressive factor released by CD8+ cells. Furthermore, it has been demonstrated that chemokine receptors cooperate in HIV entry. However, these proteins are also known to have an effect on multiple intracellular signaling cascades that may affect the process of transcription. In the present study we demonstrate that treatment of CD4+ T cells with these chemokines or with cell supernatants from HTLV-I-immortalized CD8+ T cells results in significant reduction in the abundance of HIV-1-specific RNA as analyzed by Northern blot hybridization. To examine the possibility that such suppressive factors may inhibit HIV RNA transcription, we studied the effect of RANTES, the most effective HIV-suppressive chemokine, on basal and Tat-induced HIV-directed LTR expression of a reporter gene. Neither recombinant RANTES nor conditioned medium from CD8+ cells significantly altered HIV-1 LTR-directed chloramphenicol acetyltransferase expression in either transiently or stably transfected CD4+ T cell lines, either in the presence or in the absence of Tat. These results suggest that C-C chemokines do not inhibit viral RNA transcription.

The ability of CD8 T cells derived from human immunodeficiency virus (HIV)-infected patients to produce soluble HIV-suppressive factor(s) (HIV-SF) has been suggested as an important mechanism of control of HIV infection in vivo. The C-C chemokines RANTES, MIP-1 alpha and MIP-1 beta were recently identified as the major components of the HIV-SF produced by both immortalized and primary patient CD8 T cells. Whereas they potently inhibit infection by primary and macrophage-tropic HIV-1 isolates, T-cell line-adapted viral strains tend to be insensitive to their suppressive effects. Consistent with this discrepancy, two distinct chemokine receptors, namely, CXCR4 (ref. 7) and CCR5 (ref. 8), were recently identified as potential co-receptors for T-cell line-adapted and macrophage-tropic HIV-1 isolates, respectively. Here, we demonstrate that the third hypervariable domain of the gp 120 envelope glycoprotein is a critical determinant of the susceptibility of HIV-1 to chemokines. Moreover, we show that RANTES, MIP-1 alpha and MIP-1 beta block the entry of HIV-1 into cells and that their antiviral activity is independent of pertussis toxin-sensitive signal transduction pathways mediated by chemokine receptors. The ability of the chemokines to block the early steps of HIV infection could be exploited to develop novel therapeutic approaches for AIDS.

Recently, we and others have characterized the HIV-suppressive activity of human β-defensins (hBD) 1–3. Our mechanistic studies indicate that hBD2 inhibits HIV through two distinct mechanisms. The first mechanism results in lower HIV infectivity. The second mechanism is intracellular, since it is observed even delaying treatment up to 2 hours after infection, which is not consistent with a mode of inhibition based on entry-fusion events. We hypothesized that this second antiviral mechanism is due to changes in the intracellular environment, possibly mediated by a cellular receptor. Treatment of cells in infectivity assays with an antibody specific for CCR6, a chemokine receptor that also functions as a receptor for hBD2, abrogated HIV inhibition by this defensin. Further, treatment of HIV-1 infectivity assays with MIP-3α, the cognate ligand for CCR6, resulted in inhibition of infection. Finally, single-cycle HIV infection is inhibited in cells that express CCR6 following transfection with an expression construct, but not in their CCR6low counterpart. Experiments performed using HIV pseudotyped with AMLV envelope showed that inhibition by hBD2 of HIV replication is sensitive to treatment with the Gi protein inhibitor pertussis toxin.CCR6 is an important receptor in the context of mucosal immunity, as it is expressed in CD4+CD45+CCR5+ lymphocytes, which are highly susceptible to HIV infection, and on dendritic cells, which are thought to play a crucial role in early events in HIV transmission. Therefore, an HIV suppressive mechanism targeting CCR6 is likely to play an important role in mucosal immunity to HIV.