Anthony L. DeVico and Robert C. Gallo
An inc...

Innate response
Antigen-presenting cells (dendritic cells, macrophages)

Adaptive response
Antigen-presenting cel...


One of the two main classes of T
lymphocytes (the other being
CD4 + T cells). CD8+ T cells exert
HIV-1 expression (% of control)







Patient 1

Patient 2

Patient HIV-SF



CD8+ T cell


Bulk HIV-1
supressor activity







R5 or R5X4 virus
(in a C...

Suppressor factors


Sensitive HIV phenotype

Whole PBMC




CCR5 ...


(X4 viruses
in PBMC)

(R5 viruses in

CCR5 ligands
(R5 viruses)

Unidentified CD...


A repeated sequence, several
hundred base-pairs long, found
at the two ends of the retrovir...
particularly in patients that naturally release increased
concentrations of suppressor factors. Of course, the
become highly selective antiviral agents against R5
HIV-1 strains as a consequence of their roles in an
immune res...
17. Chang, T. L.-Y., Mosoian, A., Pine, R., Klotman, M. E. &
Moore, J. P. A soluble factor(s) secreted from CD8+ T...
85. Agrawal, L., Vanhorn-Ali, Z. & Alkhatib, G. Multiple
determinants are involved in HIV coreceptor use as
154. Geiben-Lynn, R. et al. Noncytolytic inhibition of X4 virus by
bulk CD8+ cells from human immunodeficiency vir...
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Control de la infección por vih 1 por factores solubles de la respuesta inmune


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Control de la infección por vih 1 por factores solubles de la respuesta inmune

  1. 1. REVIEWS CONTROL OF HIV-1 INFECTION BY SOLUBLE FACTORS OF THE IMMUNE RESPONSE Anthony L. DeVico and Robert C. Gallo An increasing body of evidence indicates that the immune system uses a range of soluble molecules to suppress certain viral infections without killing infected host cells. Recent studies indicate that such factors might have an especially important role in the immune response to HIV-1. Accordingly, this review uses HIV-1 as a model to explore the diversity of non-cytolytic antiviral factors and considers how these molecules might be used to develop new therapeutic and prophylactic strategies to fight viral infections. NATURAL KILLER CELLS (NK cells). Lymphocytes that do not express the T-cell receptor (TCR) or B-cell receptor (BCR) and which mediate natural killing against prototypical NK-cell-sensitive targets. γδ-T CELLS T lymphocytes express a T-cell receptor (TCR) that is composed of either α- and β-subunits (αβTCR) or a TCR that is composed of γ- and δ-subunits (γδTCR). Most (>90%) T cells have an αβTCR that recognizes conventional MHC class I or II ligands. T cells expressing γδTCR are less common and the ligands of this type of receptor are less well characterized. Institute of Human Virology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202, USA. Correspondence to R.C.G. e-mail: doi:10.1038/nrmicro878 Viruses and immune systems play microscopic games of ‘hide-and-seek’ during the course of an infection. The virus attempts to find and enter its host cell, replicate its genome, assemble new particles and spread to new target cells, while minimizing its exposure to the immune system. At the same time, the immune system attempts to recognize and eliminate the invading viruses as quickly as possible and without causing damage to the host. In cases of chronic viral infections, or when viruses (such as retroviruses) use cells of the immune system as their hosts, this game can be very complicated indeed. To gain an advantage, the immune system uses overlapping effector mechanisms, which are activated by viral infection and suppress one or more steps in the virus life cycle (FIG. 1). NATURAL KILLER CELLS (NK cells)1,2, granulocytes and, possibly, γδ -T CELLS3–5 provide the initial line of defence when stimulated by chemical signals that are released at sites of infection. All of these cells produce immunoregulatory molecules: neutrophils produce antimicrobial defensins; and NK cells and γδ-T cells can kill infected cells through cytolytic mechanisms1–5. Host macrophages and dendritic cells also have important roles in this early stage by taking up and presenting viral antigens and by secreting several soluble CYTOKINES and CHEMOKINES that help to amplify the immune response. These early processes are called INNATE IMMUNE RESPONSES because they can act NATURE REVIEWS | MICROBIOLOGY immediately using extant receptors and do not require the induction of major histocompatibility complex (MHC) gene products or antigen presentation in the context of MHC molecules. Over time, the innate response gives way to an adaptive response, which requires viral antigen presentation by cell-surface MHC molecules. Adaptive immunity is carried out by several effector-cell subsets, including CD8 T CELLS, CD4 HELPER T CELLS, and B cells. These cells establish important cell–cell communication mechanisms by releasing a wide range of soluble molecules. The cellular arm of adaptive immunity is active at an early stage (~1 week post-infection) and has an important role in fighting most viral infections. CELLULAR IMMUNE RESPONSES are mediated by CD8+ cytotoxic T lymphocytes (CTLs) that have been primed by dendritic cells and other cell subsets that present viral antigens in conjunction with Class I MHC molecules. Once primed by MHC–antigen complexes and co-stimulatory signals, CTL clones target infected tissues and attempt to kill infected cells before they can produce progeny viruses. To do this, the CTL must interact with viral antigens in conjunction with Class I MHC molecules on the surface of an infected cell. The CTL then delivers pro-apoptotic signals and soluble cytolytic enzymes that destroy the infected target. Several weeks after infection, the humoral arm of adaptive immunity joins the cellular response in combating infection. HUMORAL IMMUNE RESPONSES arise as a + + VOLUME 2 | MAY 2004 | 4 0 1
  2. 2. REVIEWS Innate response Antigen-presenting cells (dendritic cells, macrophages) Adaptive response Antigen-presenting cells (dendritic cells, macrophages) Humoral Cellular γδ -T cells NK cells Cytolytic cell killing Virus CD8+ T cells Granulocytes Soluble non-cytolytic factors (IFN, TNF-α, certain CC chemokines in the case of HIV-1, other molecules) Defensin Cytolytic cell killing CD4+ T cells Soluble non-cytolytic factors (IFN, TNFα, certain CC chemokines in the case of HIV-1, other molecules) B cell Antibodies Host cell Figure 1 | A working model of an antiviral immune response that uses cytolytic cell killing, antibodies and soluble non-cytolytic factors as a means to suppress infection. In the first phase of an infection, viruses infect susceptible host cells. In response to infection, the innate immune response mediates several antiviral mechanisms, including cytolytic cell killing by NK cells, which lyse infected cells. Several soluble factors are also released, which directly suppress infection without killing infected host cells. Infected cells or virions that escape the innate response are controlled by the humoral and cellular adaptive immune responses. However, non-cytolytic antiviral mechanisms continue to have a crucial role in suppressing viral replication. Infected host cells are shown in red and uninfected cells in green. IFN, interferon; NK, natural killer; TNF, tumour necrosis factor. CYTOKINES Originally used to describe a group of immunomodulatory growth factors, the term cytokine is now used to describe a diverse group of soluble proteins that modulate the many activities of cells and tissues. CHEMOKINES Chemotactic cytokines involved in specific inflammatory responses. They are differentiated into CC or CXC chemokines on the basis of their primary amino acid sequence. INNATE IMMUNE RESPONSE The first line of defence against microbial infections. Innate responses do not require the induction of MHC gene products or antigen presentation in the context of MHC molecules. 402 | MAY 2004 | VOLUME 2 result of interactions between antigen-specific B cells and CD4+ helper T cells that have been stimulated by viral antigens in conjunction with MHC Class II antigens. Ultimately, the interacting B cells release antibodies that react with specific epitopes on the viral antigens. In most cases, antiviral antibodies prevent the intercellular transmission of virions and the reinfection of the host, although in some instances they can also contribute to the direct suppression of viral replication. Overall, the conventional view of the immune system holds that the control and prevention of viral infections rests with one or more of these defence mechanisms, depending on the virus in question. However, this view is being reconsidered. We are beginning to understand that both innate and adaptive responses to viral infections can be supplemented by NON-CYTOLYTIC SOLUBLE SUPPRESSOR FACTORS, which, remarkably, can be antiviral either by accident or design. This new perspective has emerged primarily from four lines of evidence. First, it has become clear that certain immunoregulatory cytokines, including INTERFERONS (IFNs) and tumour necrosis factor (TNF), not only induce apoptosis and necrosis of certain cell types on infection, but also activate a number of intracellular pathways that directly suppress viral replication without killing the host cell6–18. Interferons have been shown to suppress hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus, vesicular stomatitis virus (VSV), vaccinia virus, picornaviruses, retroviruses, influenza viruses and other types of viruses in vitro in a non-cytolytic manner6–13,17. These broad antiviral effects are mediated by several mechanisms that rely on receptor-mediated gene-expression pathways, including the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signal-transduction cascade6–11,18,19. In the presence of double-stranded RNA, IFN-α and IFN-β mediate well-characterized antiviral mechanisms that either degrade viral RNA transcripts or inhibit viral protein synthesis. Other mechanisms inhibit the attachment, entry or assembly of certain viruses. IFN-γ is likely to activate overlapping pathways as well as non-redundant pathways and comparable antiviral mechanisms8,11,18,19. Second, many cell subsets of both the innate and adaptive responses, including NK cells, mononuclear phagocytes, γδ-T cells, CD4+ T cells and CTLs, have been shown to secrete non-cytolytic
  3. 3. REVIEWS CD8+ T CELLS One of the two main classes of T lymphocytes (the other being CD4 + T cells). CD8+ T cells exert cytolytic activity in an antigenspecific manner. They also produce various cytokines to regulate the immune system. CD4+ HELPER T CELLS T helper cells that collaborate with antigen-presenting cells in the initiation of an immune response. CELLULAR IMMUNE RESPONSE An adaptive immune response that is mediated by CD8+ cytotoxic T lymphocytes that have been primed by dendritic cells and other cell subsets that present viral antigens in conjunction with Class I MHC molecules. Cellular responses help clear viral infections by killing infected cells. HUMORAL IMMUNE RESPONSE An adaptive immune response that is mediated by antibodies directed against specific epitopes on viral antigens. These antibodies prevent the intercellular transmission of virions and the reinfection of the host. NON-CYTOLYTIC SOLUBLE SUPPRESSOR FACTORS Diffusible molecules that are released during an innate and/or adaptive immune response and which suppress viral replication without killing host cells. INTERFERONS (IFNs). Interferons are proteins with potent antiviral activity that are of particular importance during the early response to pathogens. Type I IFNs (α, β and ω) are homologous proteins that interact with a common two-chain receptor (IFNAR1 and IFNAR2). Type II or immune IFN is represented by a single protein (IFN-γ) that interacts with different two-chain receptors (IFN-γR1 and IFN-γR2). antiviral molecules, such as IFN and TNF-α, in response to stimulation by viral antigens10. Third, a substantial amount of data from experiments with HBV transgenic mice indicate that the primary mechanism for the control and clearance of HBV infection is provided by the direct non-cytolytic antiviral activity of soluble factors that are released by CTLs and/or NK cells10,14,15. In this transgenic system, the animals exhibit hepatic HBV gene expression, but do not mount an immune response (anti-self) against HBV antigens. Furthermore, the data do not support cell–cell virus spread and reinfection by HBV. So, these animals are highly useful for specific analyses of effector subsets. Adoptive transfer experiments carried out in these mice showed that HBV-specific CTL clones suppress viral replication in many infected cells through non-cytolytic antiviral activities that are mediated by IFN-γ and, perhaps, TNF-α20–23. Fourth, cell subsets other than CTLs have been shown to release soluble factors with non-cytolytic antiviral activity. CD4+ T cells have been shown to suppress influenza, VSV, HBV and vaccinia infections in mice24–30, and HIV-1 infections in human lymphocyte cultures 31, through the release of soluble factors. NK cells have also been observed to suppress certain viral infections by releasing non-cytolytic antiviral factors32–37. The production of soluble suppressor factors by γδ-T cells has been correlated with the protection of macaques from simian immunodeficiency virus (SIV)38. Given these findings, a new model for antiviral immunity has emerged, which includes the direct antiviral effects of soluble non-cytolytic factors as an effector mechanism. In this model, the array of soluble mediator factors that are released in response to infection includes factors that diffuse through the site of infection and directly suppress virus replication without killing infected cells (FIG. 1). As will be discussed below, this working model accounts for the possibility that some of the virus suppressor molecules might also perform immunoregulatory roles. In these cases, the relative importance of antiviral versus immunoregulatory function varies depending on the infecting virus and the nature of the adaptive response. In theory, this model presents three advantages for the infected host. First, a soluble/diffusible non-cytolytic effector function would boost the antiviral potency of a CTL beyond its destructive capacity, which is inherently limited by the frequency of effector–target-cell contacts. So, a CTL would be able to more rapidly clear an infection in tissues where it is outnumbered by infected cells. Second, NK cells and CTLs could suppress infection in vital organs without having to destroy a large number of important cells. Third, an immune response to one virus might suppress other viruses in the area by bystander effects that are mediated through the diffusion of soluble antiviral factors. For example, the release of IFN by CTLs in response to one type of virus might partially ‘sterilize’ the area to other sensitive viruses. In the past such a phenomenon was difficult to show in experimental viral systems. In mice, CTL clones that are specific for lymphocytic choriomeningitis virus (LCMV) were passively transferred to animals that were then NATURE REVIEWS | MICROBIOLOGY co-challenged with LCMV and the related pichinde arenavirus. Although the animals were protected from LCMV, they were still infected by pichinde39. Similarly, passive transfer of CTLs that are specific for influenza haemagglutinin HA2 protected mice from influenza virus but not from concomitant challenge with influenza haemagglutinin HA1 (REF. 40). Such specificity indicates that if soluble non-cytolytic factors are released by antiviral CD8+ T lymphocytes, they might be active only over very short distances. On the other hand, it was shown that hepatocellular HBV gene expression was potently suppressed during LCMV infection in HBV transgenic mice41. Such suppression was noncytolytic and principally mediated by TNF-α and IFN-α/β produced by LCMV-infected intrahepatic macrophages41. Similarly, clinical studies have provided evidence that HBV replication is suppressed by acute hepatitis A virus (HAV)-induced production of soluble factors including IFN-γ 42,43. So, soluble ‘bystander’ suppression may indeed occur more readily in certain tissues and viral systems. In recent years, HIV-1 has been characterized as a virus that is highly sensitive to non-cytolytic suppression by soluble factors. Indeed, the nature of soluble HIV-1 suppressor activity might reflect an intimate link between viral replication and the immune system. So, HIV-1 infection is an ideal model for appreciating the capacities of soluble factors to mediate antiviral immunity. Accordingly, the following sections focus on HIV-1 suppressor factors, their activities and their relevance to natural infection. Soluble HIV-1 suppressor activity The first observations of non-cytolytic HIV-1 suppressor activity were made almost two decades ago in the context of CD8+ T-cell responses44–46. At that time, it was recognized that peripheral blood mononuclear cells (PBMCs) taken from seropositive asymptomatic individuals often failed to manifest HIV-1 replication in vitro. In 1986, Walker et al.44 showed that this suppression of viral replication was linked to the presence of CD8+ T cells in the cultures. Selective removal of these cells resulted in an elevation of viral replication, whereas depletion of other cell types, such as CD16+ cells (including NK cells), had no effect44. Furthermore, reconstitution of depleted cultures with autologous CD8 + T cells re-established suppression of HIV-1 replication in a concentration-dependent manner without altering the proliferation or viability of CD4+ HIV-1 host cells45–49. Taken together, these data show that CD8+ T cells are able to block active HIV-1 replication through non-cytolytic virus-suppressive mechanisms. Later studies revealed two more significant characteristics of this activity. First, the factor(s) that are responsible for HIV-1 suppressor activity are soluble45,46,50. Experiments carried out in transwell chambers clearly showed that non-cytolytic suppression was achieved even when the CD8+ T cells were separated from the CD4+ host cells by semi-permeable membranes50,51. Other experiments showed that HIV-1 suppression was mediated by filtered supernatants from VOLUME 2 | MAY 2004 | 4 0 3
  4. 4. HIV-1 expression (% of control) REVIEWS 100 80 60 40 20 0 Patient 1 Patient 2 Patient HIV-SF +Anti-RANTES +Anti-MIP-1α +Anti-MIP-1β Patient 3 Patient 4 +Anti-RANTES +Anti-MIP-1α +Ant-MIP-1β α β Figure 2 | RANTES, MIP-1α and MIP-1β are primarily responsible for the suppression of macrophage-tropic (R5) HIV-1 replication by CD8+ T-cell-culture supernatants. In this experiment, the treatment of CD8 supernatants from four HIV-positive donors with a mixture of neutralizing antibodies to RANTES, MIP-1α and MIP-1β extensively abrogates the suppression of HIV-1BaL replication. HIV-SF, HIV suppressive factors. Reproduced with permission from REF. 53 © (1995) American Association for the Advancement of Science. CD8 ANTIVIRAL FACTOR (CAF). The first reported soluble HIV-1 suppressor activity; CAF is released by primary CD8+ T cells upon activation in vitro. RANTES A CC-chemokine that binds to and activates the chemokine receptor CCR5. MIP-1α AND MIP-1β Like RANTES, MIP-1α and MIP-1β are CC-chemokines that bind to and activate the chemokine receptor CCR5. All these chemokines block the entry of HIV-1 strains that use CCR5 as a co-receptor. MACROPHAGE-TROPIC Also known as R5. A viral phenotype that is defined by the use of CCR5 as the co-receptor for viral entry. These isolates are selectively sensitive to suppression by the soluble suppressor factors RANTES, MIP-1α and MIP-1β, which are natural ligands for CCR5. T-TROPIC Also known as X4. A viral phenotype that is defined by the use of CXCR4 as a co-receptor for viral entry. These isolates are not inhibited by RANTES, MIP-1α and MIP-1β, but are sensitive to suppression by other soluble suppressor factors. 404 | MAY 2004 | VOLUME 2 cultures of CD8+ T cells that had been activated with mitogen or anti-CD3 antibody and interleukin (IL)-2 (REFS 45,51,52). Second, the soluble factor(s) are capable of suppressing many, if not all, primary HIV-1 strains46,47. CD8+ cell supernatants suppressed infection in infectivity systems that used cell-free virus stocks, as well as in experiments that used CD4+ T cells derived from HIV-positive individuals as the source of primary virus. Taken together, these findings indicate that the immunological control of HIV-1 might involve noncytolytic antiviral mechanisms, such as those shown in FIG. 1. As CD8+ T cells release the factor(s) with suppressive activity, it was reasonable to suspect that this activity is most relevant to cellular responses against HIV-1. So, the soluble suppressive factor was eventually named CD8 ANTIVIRAL FACTOR, or ‘CAF’, on the basis of the narrow assumption that activity could be explained by a single molecule. However, our perception of soluble HIV-1 suppressor activity has progressed beyond this simplistic concept in three significant ways. First, it is now appreciated that soluble HIV-1 suppressor activity reflects the collective action of multiple factors. This characteristic was revealed in 1995, when it was determined that the chemokines RANTES and macrophage inflammatory proteins 1α and 1β (MIP-1α AND MIP-1β) are involved in the suppressor activity when released by activated CD8+ T cells53. Specifically, it was shown that neutralizing anti-chemokine antibodies completely abrogate the HIV-1 suppressor activity of activated CD8+ T cells from HIV-1 seropositive, asymptomatic individuals. Notably, antibodies against any one of the chemokines had little effect on suppressor activity against the test isolate, HIV-1Bal. However, a mixture of antibodies against all three chemokines reversed the suppression of the virus (FIG. 2). This important observation showed that RANTES, MIP-1α and MIP-1β were responsible for nearly all of the HIV-1Bal suppressor activity in the CD8+ T-cell-culture supernatants. However, more extensive testing with a wider variety of isolates revealed that although RANTES, MIP-1α and MIP-1β always suppressed what were then called MACROPHAGETROPIC viruses, they did not suppress T-TROPIC isolates, such as HIV-1IIIB53. This was in contrast to unfractionated CD8+ T-cell supernatants, which suppressed all strains of HIV-1 regardless of tropism. We now know that macrophage-tropic viruses are selectively suppressed by RANTES, MIP-1α and MIP-1β owing to their specific requirements for entry. To enter target cells, HIV-1 must first establish an envelope–receptor complex that includes the viral envelope glycoprotein (gp120) and cell-surface CD4 molecule. The gp120–CD4 complex that is formed by macrophage-tropic viruses then binds selectively to a seven-transmembranespanning, G-protein-coupled surface co-receptor called CCR5 (REFS 54–58), which is also the natural receptor for RANTES, MIP-1α and MIP-1β. As a consequence of this shared receptor usage, the entry of macrophage-tropic (now called R5) HIV-1 strains is blocked by the CCR5 ligands53,59–61. Conversely, the T-tropic (now designated X4) strains enter cells using a different chemokine receptor known as CXCR4, which is not bound by CCR5 ligands58,62 but instead by the chemokine stromalcell-derived factor or SDF-1 (REF. 63). This preference renders X4 strains immune to inhibition by CCR5 ligands. So-called DUAL-TROPIC (R5X4) strains can use either co-receptor type, depending on target-cell expression patterns, but are inhibited by CCR5 ligands whenever CCR5 is the operative co-receptor. So, RANTES, MIP-1α and MIP-1β collectively account for the CD8-derived suppression of R5 viruses (and R5X4 viruses in a CCR5dependent system), whereas other, unknown factors are responsible for inhibiting the isolates that must use the CXCR4 co-receptor (FIG. 3). In riposte, ‘CAF’ is now defined as the portion of CD8-derived suppressor activity that suppresses X4 HIV-1 strains (or R5X4 strains in a CCR5-minus system), or any suppressive molecule other than RANTES, MIP-1α or MIP-1β17,64,65. In general, it is now common to categorize factors according to whether they suppress R5 versus non-R5 (X4 or R5X4) isolates. Second, it is clear that bulk CD8+ T cells are not the only human cell sources of soluble non-cytolytic HIVsuppressor activities/factors (FIG. 4). For example, naive CD4+ T cells stimulated with immobilized anti-CD3 and anti-CD28 antibodies66–68 or co-cultured with antigen-pulsed dendritic cells31, NK cells stimulated with IL-15 and IL-12 or cytokine and anti-CD16 (REFS 35–37), γδ-T cells69, and antigen-specific CD4+ T cell clones 70 have all been reported to secrete antiviral concentrations of RANTES, MIP-1α and MIP-1β together with unassigned activities that suppress R5 and/or X4 isolates. CD4- or CD8-depleted PBMCs that are stimulated by live, inactivated influenza virus71 produce HIV-suppressive levels of IFN-α together with unidentified factors that suppress both R5 and X4 HIV-1 isolates. Alloantigen-stimulated whole PBMC72 release a partially unassigned factor with antiviral activity that
  5. 5. REVIEWS CD8+ T cell RANTES Bulk HIV-1 supressor activity ? MIP-1β ? MIP-1α 'CAF' ? CCR5 R5 or R5X4 virus (in a CCR5-dependent system) X4 or R5X4 virus (in a CCR5-negative system) Host cell Host cell Figure 3 | A schematic representation of the multipartite nature of the soluble HIV-1 suppressor activity produced by CD8+ T cells. RANTES, MIP-1α and MIP-1β account for the suppression of R5 viruses. Unidentified factors suppress X4 and R5X4 HIV-1 isolates in a CCR5-negative system. CAF, CD8 antiviral factor. DUAL-TROPIC Also known as R5X4. An HIV-1 phenotype that is defined by the use of either CCR5 or CXCR4 co-receptors for viral entry. These isolates are suppressed by RANTES, MIP-1α and MIP-1β in CCR5-dependent infection systems. suppresses diverse HIV-1 strains. CTL clones that are stimulated by autologous antigen-presenting cells or anti-CD3 antibodies also release a soluble non-cytolytic factor with antiviral activity that comprises RANTES, MIP-1α, MIP-1β and unknown X4 suppressor factors73–78. These cells release RANTES, MIP-1α and MIP-1β from their cytoplasmic granules as part of a larger glycosaminoglycan (GAG) complex75. Although the immunological significance of these chemokine– GAG complexes is unclear, in the case of RANTES, MIP-1α and MIP-1β the antiviral activity is preserved after GAG binding, whereas the receptor-activating function is inhibited79. Notably, placental stromal cells were also shown to release a factor with HIV-suppressive activity80. In accordance, leukaemia inhibitory factor, which is expressed in the placenta, was shown to inhibit multiple HIV-1 strains81. Third, the composition of the suppressor factor(s) that are released in a given system might change over time. For example, four days after addition of antigen co-cultures of naive CD4+ T cells and dendritic cells31 secrete a factor with suppressive activity that comprises RANTES, MIP-1α, MIP-1β and macrophagederived chemokine (MDC), which is another HIVsuppressive chemokine82–86. The suppressive activity of the 4-day-old supernatant is almost entirely abrogated by neutralizing antibodies to these chemokines31. However, culture supernatant that is collected after stimulation is less sensitive to the neutralizing antichemokine antibodies. Furthermore, supernatants that are collected early (1 day) after addition of antigen suppress HIV-1 in a manner that is completely insensitive to these antibodies. So, the cultures release unknown inhibitors at certain time points and known suppressor factors at others. Such changes might be NATURE REVIEWS | MICROBIOLOGY explained by the differentiation of cells into distinct subsets over time. As a result, the culture supernatants reflect the presence of a dynamic collection of HIV-1 suppressive molecules, some of which have yet to be identified. Overall, these findings indicate that both innate and adaptive immune responses use soluble non-cytolytic antiviral activity to control HIV-1 replication. Findings that multiple cell subsets produce factors with antiviral activity indicate that there is a degree of redundancy in such responses. However, it seems almost certain that soluble suppressor activity is always due to the actions of multiple components (possibly providing another level of redundancy), although the nature of the composition might vary according to the cell subset and response pathway in question. The quest for other HIV-1 suppressor factors To fully understand the immunological significance and practical value of soluble antiviral activity, the responsible factors must be identified beyond RANTES, MIP-1α and MIP-1β. Accordingly, efforts have been made to assign a molecular identity to ‘CAF’ and HIV-1 suppressor activities. Early studies52,87 focused on IFN-α, -β and -γ and TNF-α, because they were already known to suppress a number of viruses (see above), including HIV-1 (REFS 52,88–96), when tested as reagents. However, two studies found that treatment of CD8+ T-cell supernatants with neutralizing antibodies against these cytokines, or other HIV-1 inhibitors, such as transforming growth factor (TGF)-β and IL-4, did not abrogate ‘CAF’ activity52,87. These studies indicated that an unknown factor is responsible for ‘CAF’ activity, but did not eliminate the possibility that ‘CAF’ is a collection of known antiviral cytokines VOLUME 2 | MAY 2004 | 4 0 5
  6. 6. REVIEWS Suppressor factors Stimuli Sensitive HIV phenotype Whole PBMC IFN-α, unidentified factors Mitogen R5 CCR5 ligands Influenza R5 Unidentified factors X4 EDN ribonuclease, unidentified factors R5 CCR5 ligands Alloantigen X4 R5 X4 NK cells Anti-CD16, cytokines Unidentified factors CD4+ T cells Antigen and dendritic cells CCR5 ligands MDC, unidentified factors CCR5 ligands X4 R5 X4 R5 Anti-CD3/ CD28 Unidentified factors CD8+ T cells Mitogen CCR5 ligands Anti-CD3 X4 Unidentified protease? CTL Antigen and antigenpresenting cells CCR5 ligands Unidentified factors R5 X4 R5 X4 Figure 4 | A variety of primary cell subsets secrete soluble HIV-1 suppressor activities in response to various stimuli. Candidate suppressor factors that are ‘relevant’ to these activities are shown. In this case, CCR5 ligands refers to RANTES, MIP-1α and MIP-1β. The HIV-1 phenotype (which is defined by co-receptor preference) that has been reported to be most sensitive to the various suppressor factors or activities is shown. CTL, cytotoxic T lymphocytes; EDN, eosinophil-derived neurotoxin; MDC, macrophage-derived chemokine; NK, natural killer; PBMC, peripheral blood mononuclear cells. with redundant functions. Indeed, a later study showed that a combination of antibodies to IL-10, IL-13, IFN-α and IFN-γ was able to appreciably reverse HIV-1 suppressor activity in CD8+ T-cell-derived supernatants97. However, the antibody mix did not achieve complete reversal of X4 HIV-1 suppression, indicating that additional unknown suppressor factors were involved. Fortunately, recent technological advances have greatly increased the chances of successfully identifying soluble HIV-1 suppressor factors. The development of herpesvirus saimiri (HVS)- and human T lymphotrophic virus (HTLV)- immortalized T-cell lines that secrete factor(s) with soluble suppressor activity53,82,98–100 has been particularly helpful because they provide continuous 406 | MAY 2004 | VOLUME 2 sources of antiviral factors, which is necessary for protein purification and sequencing efforts. More recent refinements in genomic and microanalytical techniques have facilitated direct examinations of primary cells for suppressor factors. Given these new tools, a number of additional candidate HIV-1 suppressor factors82,101–104 have been identified in recent years (FIG. 4). Nevertheless, the identification of immunologically relevant suppressor factors remains a tricky business. Many molecules, including substances present in commercial media or sera used to culture cells rather than T-cell-derived factors101, will suppress HIV-1 replication under certain conditions and/or at sufficient concentrations. Therefore, it is essential to determine if a candidate factor is produced by primary cells at effective antiviral concentrations. The most reliable method is to treat conditioned media with cognate antibodies that either neutralize biological activity or clear the native antigen from solution. Abrogation of HIV-1 suppressor activity by such treatment unambiguously shows that the candidate factor is active at the concentrations secreted by primary cells and therefore might be ‘relevant’ to a natural immune response. Of course, such analyses will not address the presence of redundant factors unless an appropriate mixture of antibodies is used. On the basis of antibody-neutralization experiments, most of the candidate human suppressor factors have been characterized as potentially relevant to soluble suppressor activity from at least one source (FIG. 4). At the same time, it is also clear that relevance is systemdependent. For example, IFN-α contributes significantly to the activity of influenza-A-stimulated PBMCs71, yet IFNs do not seem to be important components of factors with suppressor activity that are released by CD8+ T cells52,87. In the case of PBMC-derived activities, relevance is also determined by the nature of the antigenic stimulus. So, the main suppressor factor in influenza-A-stimulated PBMC cultures is IFN-α71, whereas in alloantigen-stimulated PBMC cultures104 it is a heat-stable ribonuclease known as eosinophilderived neurotoxin (EDN). This variability is perhaps not surprising given that PBMCs can contain mixtures of cell subsets with different specificities and response profiles. Overall, RANTES, MIP-1α and MIP-1β provide the most overlap among systems (FIG. 4). But the big question remains: what factors are responsible for the CD8+ T-cell-derived suppressor activity that is not explained by RANTES, MIP-1α and MIP-1β? Notably, the anti-chemokine antibody experiments (FIG. 2) indicate that these unknown factors must be significantly less potent against R5 HIV-1, as CD8 + cell-culture supernatants treated with anti-chemokine antibodies do not exhibit residual R5 suppressor activity. If these other factors were able to suppress R5 infection, their antiviral activities should have been apparent after the chemokines were neutralized, yet this was not the case. Possibly, the unknown factors are not present in the supernatants at sufficient concentrations to block R5 HIV-1 replication. On the other hand, they might need to synergize with one of the R5 ligands to mediate R5 suppression.
  7. 7. REVIEWS α-defensin MDC (X4 viruses in PBMC) MDC (R5 viruses in macrophages) CCR5 ligands (R5 viruses) Unidentified CD8+ T-cell factors ATF-uPA ? CCR4 (CCR?) CCR4 (CCR?) CD87/u-PAR STAT1? Coreceptor Reverse transcription Provirus integration Downregulate CXCR4 CD4 Attachment, fusion and entry ? EDN ribonuclease IFNAR1 IFNAR2 IFN-α,-β LTR-driven transcription Translation Assembly Budding STAT1? IFN-α,-β IFN-δR1 IFN-δR2 CD4 IFN-α,-β,-γ Crosslink IL-16 Figure 5 | Soluble factors might suppress HIV-1 at multiple replication steps through several mechanisms. Candidate factors and the replication steps they target are shown. ATF-uPa, amino-terminal peptide fragment of urokinase-type plasminogen activator; EDN, eosinophil-derived neurotoxin; IFN, interferon; IL, interleukin; LTR, long terminal repeat; MDC, macrophage-derived chemokine; STAT, signal transducer and activator of transcription; CD87/u-PAR, urokinase-type plasminogen activator receptor. Of course, these possibilities cannot be explored until the unknown factors have been identified. Early on, the CXCR4 ligand SDF-1 was considered to be a logical candidate for ‘CAF’ when it was determined that it binds to CXCR4 co-receptors and blocks the entry of both X4 and dual-tropic viruses in CCR5minus systems63. But lymphocytes produce little or no SDF-1 and these levels do not correlate with CD8+ cellderived suppressor activity105. Another early candidate for ‘CAF’ was the cytokine IL-16. This cytokine is constitutively produced by CD4+ and CD8+ T cells106, and inhibits HIV-1 replication in vitro when tested as a recombinant molecule103,106–109. However, IL-16 is not considered relevant to ‘CAF’ as it was shown that the concentrations of cytokine that are released by CD8+ cells do not correlate with levels of HIV-1 suppressor activity in culture supernatants. Furthermore, neutralizing anti-IL-16 antibodies do not reverse the soluble HIV-1 suppressor activities that are derived from CD8+ cell cultures107. Other reports have suggested that ‘CAF’ activity might be attributed to a fragment of bovine antithrombin III101, or to a catalytically inactive amino-terminal peptide fragment of urokinase-type plasminogen activator (ATF-uPA)110,111. These possibilities remain to be explored. Recently, Ho and colleagues attributed ‘CAF’ activity to the α-defensins 1, 2 and 3 (REF. 97). Defensins are well known as neutrophilderived antibiotic factors and others had already observed that they inactivate certain enveloped viruses112–114, including HIV-1 (REF. 115). However, the assertion that ‘CAF’ is explained by a combination of RANTES, MIP-1α, MIP-1β and α−defensins102, has not ‘stood the test of time’. Following evidence that the antiviral properties of ‘CAF’ and α-defensins are discordant116–119, the Ho group revealed that the α-defensins NATURE REVIEWS | MICROBIOLOGY present in their cultures were released by contaminating neutrophils and not by CD8+ T cells120, and therefore could not contribute to ‘CAF’. So, the search for a specific CD8+ T-cell-derived HIV-1 suppressor factor that is active against X4 viruses continues. Although the nature of the unknown CD8-derived factors remains obscure, there are tantalizing indications that protease activity is involved. A recent study showed that certain protease inhibitors abrogate the suppressor activity in CD8+ T-cell supernatants that act on X4 isolates121. In accordance, it was suggested that the HIV-suppressive fragment of bovine antithrombin III is generated by a proteolytic activity that is greater in CD8+ T-cell supernatants from HIV-infected donors than from seronegative donors101. Of course, this protease has no clinical relevance until it is shown to process human substrate into an antiviral form. Mechanisms of non-cytolytic HIV-1 suppression Soluble suppressor activities suppress many HIV-1 strains. Such breadth of activity is consistent with the presence of multiple suppressor mechanisms, which could selectively become activated according to viral phenotype and tropism (FIG. 5). In accordance with this view, suppressor factors collectively exhibit a diverse range of antiviral mechanisms. MDC inhibits the replication of R5 viruses in macrophages, but does not interfere in proviral DNA accumulation84. However, MDC suppresses X4 viruses at the level of reverse transcription in primary T cells. The X4-suppressive effects involve a G-protein-coupled signalling pathway (C. Kleinman, A.L.D. and A. Garzino-Demo, unpublished observations) that is linked either to the MDC receptor CCR4 or to an unidentified receptor that might be used by a naturally truncated form of the chemokine83. EDN VOLUME 2 | MAY 2004 | 4 0 7
  8. 8. REVIEWS LONG TERMINAL REPEAT A repeated sequence, several hundred base-pairs long, found at the two ends of the retroviral genome. LONG-TERM NON-PROGRESSORS HIV-infected persons who remain clinically healthy for long periods of time in the absence of antiretroviral therapy. These persons seem to have a natural capacity to effectively control HIV-1 infection. 408 | MAY 2004 | VOLUME 2 ribonuclease is associated with a mechanism that blocks replication prior to reverse transcription72,104,122. The exact mechanism of suppression is not known, but it might be similar to the RNase-dependent pathways that are induced by IFNs6–11. IFN-α also blocks HIV-1 replication prior to reverse transcription71, in part due to the downregulation of the CXCR4 co-receptor12,13, and also at the budding and assembly steps123. ATF-uPA also interferes with the budding and assembly steps of HIV-1 replication through receptor-mediated pathways110,111. The α-defensins also suppress HIV-1 by altering the host-cell environment116, although they can directly bind and inactivate other viruses113. In the case of HIV-1, suppression occurs at a post-entry step that might be as late as proviral integration116. IL-16 is an interesting case because it is a ligand for CD4 (REF. 106). However, this binding does not prevent virus attachment or gp120 interactions. Instead, CD4 crosslinking by IL-16 homodimers or homotetramers generates several second messengers that ultimately inhibit HIV-1 gene transcription106,108 and, possibly, the entry of virions into macrophages and dendritic cells109. Notably, IL-16 has also been shown to suppress HIV-1 replication, even when present intracellularly124. As discussed above, RANTES, MIP-1α and MIP-1β suppress the entry of R5 isolates; details of this process have been exhaustively covered in several excellent reviews (for example, REF. 58) and need not be described further. However, it should be noted that three other CCR5 ligand — monocyte chemoattractant protein (MCP)2 (REF. 59), LD78β60 and a natural fragment of human β-CC-chemokine (HCC)-1 (REF. 61) — also suppress R5 HIV-1 strains at the level of entry, although these molecules have not yet been formally linked to a soluble suppressor activity. Crude CD8+ T-cell supernatants contain yet another antiviral activity that has not been assigned to any known factor. This activity blocks HIV-1 LONG TERMINAL 17,116,125–131 REPEAT (LTR)-driven transcription , presumably through signal-transduction pathways that are modulated by receptor–ligand interactions. However, human CD8+ cell supernatants also suppress transcription that is driven by other retroviral promoters127, which indicates that the responsible antiviral agent(s) is not specific to lentiviruses. In vitro assays indicate that nuclear factor (NF)-κB, nuclear factor of activated T cells (NFAT), and STAT1 might be involved in the suppressive mechanism17,127,128, although suppression of a SIV mutant was observed in the absence of a NF-κB binding domain in the LTR131. The inhibition of LTR-driven transcription is reminiscent of HIV-1 suppression by IL-16, IFN-β, IFN-γ and TGF; however, as discussed above, these molecules do not apparently explain the CD8-derived activity. Therefore, it is still assumed that a single unknown factor that is released by CD8+ cells mediates the transcriptional block. However, in the spirit of Ephraim Racker’s tenet “Do not waste clean thinking on dirty enzymes”, caution should be exercised when assigning functional mechanisms to crude material. It is possible that the antiviral effects of crude CD8+ cell medium, which is the definition of CAF, reflect the collective action of several factors, which might act in an indirect manner — for example, cause the release of IFN and IL-16. It is also unclear how this mechanism would relate to the apparent link between CD8-derived HIV-1 suppression and protease activity (see above). Relevance of HIV-1 suppressor factors The studies discussed in the previous section imply that, at least in some viral infections, soluble noncytolytic antiviral factors might come from multiple sources, under multiple conditions and target multiple steps in the virus life cycle. Given this redundancy, it seems possible that soluble suppressor factors are able to produce a barrier web that would make the average spider blush. But is there any evidence that such a web is catching bugs in vivo? The answer to this question is found in the clinic. The first evidence that non-cytolytic activities might be clinically relevant was provided by studies that showed that CD8+ T cells from asymptomatic HIV-1-infected individuals suppress HIV-1 replication more efficiently than cells from patients with AIDS45,46,133–135. Similarly, lymphoid tissue CD8+ T cells from infected persons who remain clinically healthy for extended periods of time without clinical intervention (known as LONG-TERM NON-PROGRESSORS) were shown to mediate more potent suppression than cells from AIDS patients135. More recent studies have focused on candidate factors rather than gross suppressor activity. Numerous clinical studies have compared the concentrations of RANTES, MIP-1α or MIP-1β released from PBMCs and CD8+ T cells in vitro with the clinical status of the cell donor136–141. These studies have repeatedly shown a statistically significant correlation between increased production of MIP-1α and/or MIP-1β from activated cells in vitro and favourable clinical profiles, such as increased CD4+ T-cell counts or reduced viral loads. In accordance, genetic analyses have uncovered polymorphisms in the human RANTES promoter that increase mRNA transcription and also correlate with slower HIV-1 disease progression139,142–145. Notably, a recent study found that HIV-infected patients who controlled their infection better after structured interruption of therapy harboured R5 viruses that were significantly more susceptible to suppression by RANTES146. These data support the concept that soluble noncytolytic antiviral activities dampen HIV-1 infection in vivo, although mainly in patients with an intrinsic capacity to release high levels of suppressor factors. This proposal is consistent with the general model for soluble non-cytolytic antiviral responses that is shown in FIG. 1. However, it is important to recognize that concentrations of CCR5 ligands and/or ‘CAF’ correlate with disease status even when non-HIV-1 antigens or mitogens are used to stimulate the cells45,46,133–135,137–141. Furthermore, studies on CTLs derived from infected patients showed that the release of HIV-1 suppressor factors is not restricted to HIV-1-specific cells77. So, the activation of the immune system might be sufficient to suppress HIV-1 through soluble mechanisms,
  9. 9. REVIEWS particularly in patients that naturally release increased concentrations of suppressor factors. Of course, the magnitude of this ‘bystander’ suppressive effect would depend on the nature of the antigenic stimulus and the frequencies of cognate responder cells that release suppressor factors. The ratio of naive to memory cells might also have an effect as memory cells should rapidly release soluble suppressor factors by degranulation, whereas naive cells produce them more slowly de novo. The available data further indicates that an intrinsic capacity to secrete high concentrations of HIV-1 suppressor factors might afford a certain level of resistance to primary HIV-1 infection. Lymphocytes from HIV-1 seronegative people produce variable levels of soluble HIV-1 suppressor activity following stimulation with mitogen or other non-HIV-1 antigens31,47,71,72,98,104,122,140,141,147–155. Adult individuals with lymphocytes that release abnormally high concentrations of RANTES, MIP-1α or MIP-1β seem to remain uninfected despite repeated exposure to HIV-1 (REFS 150,151). Uninfected children born to HIV-1-positive mothers exhibited the same trait 152. This intrinsic capacity might be genetic or acquired owing to previous immunological perturbations. There is also evidence that certain microorganisms elicit immune responses that dampen clinical HIV-1 infection via soluble non-cytolytic suppressor factors. The situation seems analogous to what has been reported for HBV suppression during LCMV infection in transgenic mice41 or acute HAV infection in humans42,43. Some individuals who are simultaneously infected with HIV-1 and either dengue157, Orientia tsutsugamushi (the causative agent of scrub typhus)157–159, hepatitis G/GB virus C160–164 and measles morbillivirus165 have reduced HIV-1 viral loads and/or increased CD4+ T-cell counts compared with patients who are infected only with HIV-1 or with HIV-1 and other pathogens. As passive transfer of cell-free plasma from donors with mild O. tsutsugamushi infection has been shown to reduce viral loads in HIV-1-infected recipients and suppress HIV-1 replication in vitro158, a soluble factor is responsible for the effect. The activity has not been linked to HIV-reactive antibodies. Furthermore, in vitro stimulation of PBMCs with O. tsutsugamushi induced the production of RANTES and generated resistance to R5, but not X4 or R5X4, HIV-1 infection159. In the case of hepatitis G virus (HGV), binding of the serum HGV E2 protein to CD81 led to increased RANTES secretion and reduced CCR5 expression164. Notably, both dengue virus and HGV are flaviviruses and therefore might induce the release of suppressor factors through similar immunological mechanisms. A prospective study of commercial sex workers in Senegal showed that HIV-2 infection reduces the risk of HIV-1 infection166. Stimulated cells from these persons secreted abnormally high concentrations of RANTES, MIP-1α and MIP-1β and resisted infection by R5 HIV-1 strains166). In vitro studies indicate that HTLV-II infection might upregulate the production of MIP-1α167. Given these findings, it can be envisioned that some NATURE REVIEWS | MICROBIOLOGY microbial infections efficiently elicit the secretion of soluble HIV-1 suppressor factors at antiviral concentrations within reach of HIV-1 replication sites (FIG. 1). These concepts warrant further experimental evaluation. On the other hand, there are certain clinical observations that remain difficult to reconcile with the concept of soluble suppressor activity as a mechanism of immunity. The infection of CD4+ T cells by HIV-1 is an example. As discussed above, naive and antigenstimulated CD4+ subsets release soluble suppressor factors. Yet one report suggests that the HIV-1-specific CD4+ T-cell population contains more viral DNA than other memory CD4+ T-cell pools168. It is possible that the close and prolonged proximity of these cells to actively replicating HIV-1 overcomes the suppressive capacities of soluble factors. Nevertheless, it is important to recognize that most antigen-responsive HIV-1-specific CD4+ T cells are not infected by HIV-1 (REF. 168), despite their increased chances of exposure to virus. This leaves the possibility that soluble factors provide resistance to a fraction of cells under certain conditions. Likewise, a subset of CD3– CD56+ CD4+ NK cells is persistently infected with HIV-1 in vivo even though bulk CD56+ NK cells release soluble suppressor factors, including RANTES, MIP-1α and MIP-1β169. However, other NK-cell subsets might be protected by soluble suppressor activity given the resistance of the bulk NK cell population to HIV-1 infection in vitro35–37. In other words, NK cells might represent a case of selective resistance to infection according to viral phenotype. In any case, it seems clear that the impact of soluble suppressor factors in vitro will depend on a variety of parameters, including duration and proximity to infection, viral replication kinetics versus the kinetics of production and uptake, and local viral phenotype. Practical applications of suppressor factors In view of the available evidence, it is logical to consider soluble non-cytolytic suppressor activity as a model for developing therapeutic and prophylactic antiviral agents. Some measure of support for this proposal has been provided by HBV vaccine studies carried out in both mice and humans. DNA-based HBV vaccines were shown to control viral replication in HBV-transgenic mice through the release of soluble non-cytolytic factors from CD4+ and CD8+ lymphocytes170–172. Although therapeutic HBV vaccination of humans has not provided significant clinical benefit, in some studies it was associated with reduced serum HBV DNA levels and enhanced T-cell proliferative responses that produced high concentrations of TNF-α and IFN-γ171,172. The direct administration of IFN-α is a successful treatment for HCV infection. In the case of HIV-1 infection, the arguments for exploiting soluble suppressor factors are obvious. The factors are non-toxic to target cells and, unlike antibodies or CTLs, they suppress diverse HIV-1 strains. Moreover, a significant portion of the bulk antiviral activity of primary cells is derived from factors that suppress HIV-1 as a consequence of normal physiological functions. So, RANTES, MIP-1α and MIP-1β VOLUME 2 | MAY 2004 | 4 0 9
  10. 10. REVIEWS become highly selective antiviral agents against R5 HIV-1 strains as a consequence of their roles in an immune response. One approach towards exploiting these features is to design antiretroviral drugs that mimic the activities of soluble suppressor factors. Indeed, entry inhibitors that target CCR5 are likely to represent the most promising category of new antiretroviral agents. Another approach might be to deliberately modulate the immune system to enhance the release of soluble suppressor factors from effector cells. A recent study showed that the bacterial G1 cytostatic agent rapamycin causes PBMCs to secrete increased concentrations of RANTES, MIP-1α and MIP-1β173 and to downregulate the CCR5 co-receptor. This effect rendered PBMCs and macrophages resistant to infection by R5 strains. Similarly, it was shown that cells treated with other agents that inhibit the G1 phase of the cell cycle, such as hydroxyurea, secreted increased concentrations of soluble HIV-1 suppressor factors, particularly RANTES, MIP-1α and MIP-1β174. Rapamycin has been used in humans to treat transplant rejection and hydroxyurea has been tested in clinical trials in combination with anti-HIV-1 drugs. Therefore, these compounds might provide a feasible and expedient means to clinically evaluate this concept. Furthermore, these agents might be used in combination with CCR5-targeted drugs to produce a potent, synergistic antiviral effect. Going a step further, a few research groups, including our own, have proposed the concept of using HIV-1 vaccines to enhance the capacity of the immune system to release soluble HIV-1 suppressor activity together with classical immune responses. Fortunately, primate models can be used to evaluate this concept as CD8+ T cells from chimpanzees, baboons and macaques secrete a primate equivalent of ‘CAF’ that suppresses HIV-1, HIV-2 and SIV, respectively175–177. So far, primate vaccine strategies that are based on non-pathogenic strains, live attenuated viruses or SIV antigens delivered to iliac lymph nodes have associated protection from virus challenge with the enhanced cellular capacity to release CD8-derived soluble suppressor factors, particularly RANTES, MIP-1α and MIP-1β38,178–184. The aim is to translate these findings into a vaccine strategy that might be used in humans. Lehner and co-workers showed that allo-immunization of women 1. 2. 3. 4. 5. 410 Biron, C. A., Nguyen, K. B., Pien, G. C., Cousens, L. P. & Salazar-Mather, T. P. Natural killer cells in antiviral defense: function and regulation by innate cytokines. Annu. Rev. Immunol. 17, 189–220 (1999). Biron, C. A. & Brossay, L. NK cells and NKT cells in innate defence against viral infections. Curr. Opin. Immunol. 13, 458–464 (2001). Wallace, M., Malkovsky, M. & Carding, S. R. γ/δ T lymphocytes in viral infections. J. Leukoc. Biol. 58, 277–283 (1995). Welsh, R. M. et al. α β and γ δ T-cell networks and their roles in natural resistance to viral infections. Immunol. Rev. 159, 79–93 (1997). Selin, L. K., Santolucito, P. A., Pinto, A. K., Szomolanyi-Tsuda, E. & Welsh, R. M. Innate immunity to viruses: control of vaccinia virus infection by γ δ T cells. J. Immunol. 166, 6784–6794 (2001). | MAY 2004 | VOLUME 2 6. (to prevent spontaneous recurrent abortion) caused cells to upregulate the release of CD8-derived soluble suppressor factors and CCR5 ligands. In addition, cells from these women became less susceptible to infection by HIV-1 and exhibited lower frequencies of co-receptor expression185. These provocative results indicate that a prophylactic level of soluble factor release might be achieved with a vaccine that does not necessarily contain HIV-1 antigens. Concluding remarks Viruses have evolved replication processes that allow them to coexist with the immune systems of their hosts. So, no single viral system can be used to fully define ‘antiviral immunity’. It is only through studies of different viral systems that we can fully appreciate the capacity of the immune system to mediate antiviral immunity. Research on primate lentiretroviruses and other types of viruses, such as HBV, have indicated that soluble non-cytolytic activity might provide an important mechanism for controlling at least some viral infections. Yet it is also possible that the non-cytolytic suppression of antigen production might provide a virus with a method to persist in its host. Indeed, some viruses might have evolved to coexist with such suppression as it does not immediately kill the virus or the host. On the other hand, there are likely to be some viral infections that do not involve soluble non-cytolytic factors at all. Nevertheless, it is entirely reasonable to view the soluble non-cytolytic antiviral activity as a promising basis for developing treatments for viral infections. In the case of HIV-1 infection, it has become evident that soluble suppressor activity is mediated by diverse arrays of molecules that are produced by a variety of cell subsets. This reality may not be as captivating as the concept of a single ‘magic bullet’ factor that suppresses HIV-1 in multiple settings, but the possibilities that are provided by this diversity significantly expand the potential for developing therapeutic or prophylactic antiviral strategies based on the mechanisms revealed by soluble, non-cytolytic immunity. 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Online links DATABASES The following terms in this article are linked online to: Entrez: hepatitis A virus | hepatitis B virus | HIV-1 | human T lymphotropic virus | simian immunodeficiency virus | vaccinia virus | vesicular stomatitis virus LocusLink: CCR5 | CXCR4 | IFNs | TNF SwissProt: eosinophil-derived neurotoxin | MIP-1α | MIP-1β FURTHER INFORMATION Access to this interactive links box is free online. VOLUME 2 | MAY 2004 | 4 1 3