Innate Immune System Damage in Human Immunodeficiency Virus Type 1 Infection Implications for Acquired Immunity and Vaccine Design SARAH HOWIE, ROBERT RAMAGE, and TIM HEWSON Immunobiology Unit, MRC Centre for Inflammation, and Departments of Pathology and Chemistry, Edinburgh University, Edinburgh, United Kingdom
HIV infection affects the innate as well as the acquired immune systems. Critically, it changes the function of macrophages, which link the innate and acquired responses through their ability to present antigen to CD4⫹ T lymphocytes. Patients with HIV infection have a reduced capacity to deal with subsequent pathogen exposure and many suffer from chronic pulmonary infections. We have produced complex synthetic peptides that mimic the function of viral gp120 and may represent prototypes of molecules that can prevent or ameliorate HIV-induced damage to the immune system.
(TNF) promoter associated with differential rates of disease progression (13, 14); a G-to-A substitution in the promoter of the gene encoding the ␣-chemokine stromal cell-derived factor (SDF-1), leading to accelerated progression to AIDS (15); mutations in -chemokine receptors, which are associated with protection from infection and long-term nonprogression to AIDS (16).
Since human immunodeficiency virus (HIV) infection was discovered in the 1980s the virus has infected in excess of 48 million people worldwide (1% of the world’s sexually active population). Of those infected more than 16 million have died. This represents a global health and socioeconomic problem as the number of infections is increasing yearly and predominantly in the “young adult” section of society (1).
During infection of cells gp120 in the viral envelope binds to CD4 and a chemokine receptor on the target cell surface. The CD4-binding site (17) includes several key conserved residues in gp120. The chemokine receptor-binding site depends both on the sequence of the V3 loop and on conserved residues in the gp120 molecule (18, 19). The conformation of the V3 loop changes on CD4 binding, which may allow previously hidden conserved residues access to chemokine receptors. Because of a differential distribution of chemokine receptors between cell types, the receptor to which a particular gp120 is able to bind influences the cellular tropism of the virion. Most primary isolates are macrophage (M) tropic, can infect both macrophages and T cells, and use the CCR5 coreceptor. Laboratory-adapted strains grown for many passages in T cell lines are only T cell (T) tropic and use CXCR4 as a coreceptor. The CCR5 receptor binds the -chemokines macrophage inflammatory protein 1␣ (MIP-1␣), MIP-1, and RANTES (regulation on activation, normal T cell expressed and secreted) and CXCR4 is the receptor for SDF-1 (reviewed in Reference 2). The sequence of the V3 loop mutates during the course of infection within an individual and a shift from an M- to a T-tropic virus population is associated with disease progression (20). The outcome of infection is complicated and likely to be affected by microenvironmental factors as some macrophagederived cytokines, especially those that are proinflammatory (TNF, interleukin 1 [IL-1], and IL-6), upregulate viral replication in infected cells, whereas others (IL-4, IL-10, and interferon  [IFN-]) downregulate HIV-1 production (21). In contrast to T cells, nondividing, resting macrophages can become productively infected (22). However, macrophages produce HIV-1 at a slower rate than do activated T cells. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been implicated in some of the signals controlling HIV-1 production by APCs (23). GM-CSF enhances virus production in primary monocytes and macrophages, but not in T cells or Langerhans cells (23). GM-CSF alone promotes the differentiation of monocytes to macrophages and together with IL-4 drives monocytes to become dendritic cells. Differentiation of monocytes down either of these pathways appears to result in the downregulation of surface CD14 (24), a lipopolysaccharide (LPS)/bacterial lipoprotein receptor (25). Infected macrophages stimulated with LPS (as might be expected to happen in vivo in an infected individual) show upregulation of HIV-1 expression that is abrogated by anti-CD14 antibodies (26).
HIV INFECTION AND THE INNATE IMMUNE SYSTEM Although there are two subspecies of HIV most infection and disease are due to HIV-1. The most obvious immunological problem caused by the virus is the gradual loss of CD4⫹ T lymphocytes, but there are a number of other immunological abnormalities, including a wide range of defects in the innate immune system (reviewed in Reference 2). The abnormalities in innate immune function contribute to the lung pathology associated with HIV infection (3). These include downregulation (4) of the normally continuous (5) NO production by the lung epithelium, upregulation of NO production by tissue macrophages (6), abnormalities in systemic (7) and pulmonary (8, 9) glutathione metabolism, and alterations of macrophage function (reviewed in Reference 2). Effects of HIV-1 on macrophages are of particular immunological importance as these cells link the innate and acquired immune systems via their ability to act as antigen-presenting cells (APCs). APC function is also dysregulated in HIV-1 infection (reviewed in Reference 2). Thus HIV-induced defects in macrophage function have profound effects for both innate and specific immune resistance to pathogens in infected patients. In addition to altering healthy innate immune mechanisms, certain primary defects in innate immunity predispose patients to a worse outcome with HIV-1 infection. These include null alleles of C4 associated with poor antibody responses to HIV-1 and rapid progression (10, 11); homozygosity for loci conferring low serum levels of mannose-binding protein (MBP) associated with increased HIV susceptibility and faster disease progression (12); polymorphisms of the tumor necrosis factor Correspondence and requests for reprints should be addressed to S. Howie, Ph.D., Department of Pathology, Edinburgh University Medical School, Teviot Place, Edinburgh EH8 9AG, UK. E-mail:
[email protected] Am J Respir Crit Care Med Vol 162. pp S141–S145, 2000 Internet address: www.atsjournals.org
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the Th1-promoting cytokine IL-12 in response to gp120 if they had been previously primed by IFN- (30). Th2 and Th0 cells are able to replicate HIV-1 more efficiently than Th1 cells (31, 32). The differentiation of Th0 cells is controlled by the cytokine microenvironment provided by the APCs that activate them. Of APC-derived cytokines, IL-4 upregulates, and IL-12 downregulates CXCR4 expression and therefore infectability by the T cell-tropic HIV-1 strains associated with disease progression (33, 34). This might argue for a Th2 (IL-4) shift increasing selective pressure on the virus to use CXCR4. However, another Th2 cytokine, IL-10, increases CCR5 (35) expression, and so the picture is complex and far from clear. Work in our laboratory (36) has shown that recombinant gp120 induces substantial loss of CD4 from the surface of cultured primary macrophages. This loss was observed only with gp120 derived from an mRNA sequence from an M-tropic isolate. This strain specificity, together with the failure to observe CD4 loss in ccr5 null macrophages, suggests the involvement of the CCR5 chemokine receptor in gp120-induced CD4 downregulation on antigen-presenting cells.
LOSS OF APC NUMBERS IN HIV-1 DISEASE
Figure 1. Schematic representation of peptide 3.7. Boxed residues (D368, E-370, W-427, and D-457) are critical for CD4 binding. Circled residues (K-421 and Q-422) are involved in M-tropism.
HIV-1 gp120 ALTERS APC FUNCTION Once established in the body, HIV-1 is able to infect a range of cell types (reviewed in Reference 27), but it may also disrupt the function of uninfected immune cells through interactions of soluble gp120 with its cell surface receptors on APCs and lymphocytes. gp120 alone induces interferon (mainly ␣ with some ␥) production (28, 29). As monocytes differentiate into macrophages they show enhanced IFN- production in response to HIV-1 infection or soluble gp120 alone. IL-10 secretion in response to gp120 alone was also observed and could cause the switch from helper T cell types 1 (Th1) to Th2 responses observed in HIV-1 disease (30). In contrast, macrophages produced only
HIV-1 disease leads to a reduction in the number of APCs in the periphery (losses in skin, blood, and gut have all been described) (37). The loss of cells may be due to lysis of infected APCs by cytotoxic T lymphocytes (37). Antibodies to cellular proteins and a wide range of autoimmune disorders have been detected in HIV-1 infection (38). A reduction in Langerhans cell numbers in skin could be due to a failure of hematopoiesis or a failure of tissue colonization by cells from bone marrow progenitors. Knight and Patterson (37) report that CD34⫹ bone marrow-derived stem cells show little capacity to develop (morphologically or functionally) into dendritic cells (DCs) in patients with advanced acquired immunodeficiency syndrome (AIDS).
DAMAGE DONE TO THE ACQUIRED IMMUNE SYSTEM BY APCs IN THE PRESENCE OF HIV-1 HIV-1-infected APCs or APCs that have interacted with HIV-1 proteins show a reduced capacity to stimulate T cell effector function and can prime T cells for activation-induced cell death (AICD) by apoptosis. The decline in stimulatory capacity
Figure 2. Colocalization of CD4 (i) and peptide 3.7 (ii) on CD4⫹ MM6 cells. Confocal laser scanning photomicrographs at ⫻1,600 (original magnification). (Reproduced from Howie, S. E. M., M. L. Fernandes, I. Heslop, T. J. Hewson, G. J. Cotton, M. J. Moore, D. Innes, R. Ramage, and D. J. Harrison. FASEB J. 1999;13:503–511.)
Howie, Ramage, and Hewson: Innate Immune System Damage in HIV-1 Infection
Figure 3. Inhibition of MIP-1␣ binding to MM6 cells by peptide 3.7, detected by flow cytometry. Both peptide 3.7 and an irrelevant peptide (FMDV) were used at 0.1 mM. (Reproduced from Howie, S. E. M., M. L. Fernandes, I. Heslop, T. J. Hewson, G. J. Cotton, M. J. Moore, D. Innes, R. Ramage, and D. J. Harrison. FASEB J. 1999;13:503–511.)
probably results from the loss of immunologically important surface molecules by APCs (39), including alveolar macrophages (40), seen in HIV infection. The induction of AICD in T cells is probably dependent on expression of Fas ligand at the APC membrane, which is induced by HIV infection (41, 42).
RATIONAL DESIGN OF A gp120 PEPTIDE TO BLOCK CD4 AND CCR5 BINDING OF HIV-1 A vaccine against transmitted HIV-1 would ideally prevent binding of macrophage (M)-tropic gp120 to CD4 and to the CCR5 -chemokine binding coreceptor. A vaccine peptide to prevent primary infection would thus minimally contain conserved regions of the gp120 CD4- and -chemokine receptorbinding sites. On the basis of the peptide sequence of conserved
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regions of gp120 we synthesized a series of novel peptides from sequence discontinuous but structurally contiguous regions of gp120 known to be involved in CD4 binding (43–45). At the time nothing was known that the structure of the chemokine receptor-binding site, but Lys-421 and Gln-422 were known to be involved in M-tropism (46) and are close to Trp-427, which is part of the CD4-binding site. One of the peptides (referred to as peptide 3.7) was thus synthesized to contain four residues necessary for CD4 binding (Asp-368 and Glu-370 from C3; Trp-427 and Asp-457 from C4) (46, 47); an oxidized Cys–Cys turn based on the disulfide link between Cys-378 and Cys-445; and Lys-421 and Gln-422. Peptide 3.7 has a unique structure that could not be reproduced by conventional genetic engineering (Figure 1). Peptide 3.7 contains recognized human cytotoxic T lymphocyte epitopes and human and murine antibody epitopes (48) and cross-reacts with polyclonal sheep antibody raised against recombinant gp120, indicating that it contains epitopes present in the native molecule (45). Peptide 3.7 colocalizes with CD4 on the cell surface of the human MM6 macrophage cell line (Figure 2) and like native gp120 binds to the CDR2 region of domain 1 of CD4 (45). Significantly, the peptide also inhibits MIP-1␣ binding to MM6 cells (Figure 3), indicating that it binds to both CD4 and -chemokine receptor. The peptide is also biologically functional and can inhibit the infection of primary human peripheral blood-derived macrophages by M-tropic HIV-1 (Figure 4). Like gp120, peptide 3.7 did inhibit binding of MIP-1␣ to MM6 cells, suggesting that the peptide may adopt a structure that allows it to bind to -chemokine receptors as well as CD4, or that its binding to CD4 causes either a steric alteration or a downregulation of MIP-1␣ receptors, which inhibits binding of the natural ligand. There are a number of mechanisms by which peptide 3.7 may be inhibiting MIP-1␣; first, peptide 3.7 may induce a conformational change in CD4 that causes CD4 to associate with the MIP-1␣ receptor and hence allosterically
Figure 4. Peptide 3.7 inhibits HIV-1 infection of primary human macrophages. (A) RT-PCR detection of HIV-1 BAL and -actin transcripts 72 h postinfection of blood monocyte-derived macrophages. Lane 1, markers; lane 2, macrophages preincubated with peptide 3.7; lane 3, macrophages preincubated with irrelevant peptide; lane 4, macrophages preincubated with gp120. (B) Relative band intensities in lanes 2, 3, and 4 of HIV-1, compared with -actin, 36 h postinfection. (Reproduced by permission from Howie, S. E. M., M. L. Fernandes, I. Heslop, T. J. Hewson, G. J. Cotton, M. J. Moore, D. Innes, R. Ramage, and D. J. Harrison. FASEB J. 1999;13:503–511.)
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occlude the MIP-1␣-binding site; second, a single molecule of peptide 3.7 may bind to both CD4 and the MIP-1␣ receptor simultaneously; third, separate molecules of peptide 3.7 may be binding to MIP-1␣ receptor and CD4. That the synthetic peptide 3.7 derived from three discontinuous sequence stretches of conserved regions can adopt a structure that allows it to interact with cell surface ligands of native gp120 has implications for the development of both therapeutic intervention and a synthetic vaccine. Derivatives of such a compound could provide therapeutic agents for preventing or mitigating damage to both the innate and acquired immune systems caused by HIV infection. References 1. World Health Organisation statistics: http://www.who.int 2. Hewson, T., N. Lone, M. Moore, and S. Howie. 1999. Interactions of HIV-1 with antigen-presenting cells. Immunol. Cell Biol. 77:289–303. 3. Agostini, C., and G. Semenzato. 1996. Immunologic effects of HIV in the lung. Clin. Chest Med. 17:633–645. 4. Loveless, M. O., C. R. Phillips, G. D. Giraud, and W. E. Holden. 1997. Decreased exhaled nitric oxide in subjects with HIV infection. Thorax 52:185–186. 5. Guo, F. H., H. R. Deraeve, T. W. Rice, D. J. Stuehr, F. M. Thunnissen, and S. C. Erzurum. 1995. Continuous nitric-oxide synthesis by inducible nitric-oxide synthase in normal human airway epithelium in-vivo. Proc. Natl. Acad. Sci. U.S.A. 92:7809–7813. 6. Hori, K., P. R. Burd, K. Furuke, J. Kutza, K. A. Weih, and K. A. Clouse. 1999. Human immunodeficiency virus-1-infected macrophages induce inducible nitric oxide synthase and nitric oxide (NO) production in astrocytes: astrocytic NO as a possible mediator of neural damage in acquired immunodeficiency syndrome. Blood 93:1843–1850. 7. Jahoor, F., A Jackson, B. Gazzard, G. Philips, D. Sharpstone, M. E. Frazer, and W. Heird. 1999. Erythrocyte glutathione deficiency in symptomfree HIV infection is associated with decreased synthesis rate. Am. J. Physiol. Endocrinol. Metab. 39:E205–E211. 8. Adams, J. D., G. S. Jaresko, S. G. Louie, L. K. Klaidman, D. Kennedy, O. Sharma, and C. T. Boylen. 1993. Pneumocystis-carinii pneumonia in HIV-infected patients—effects of the diseases on glutathione and glutathione disulfide. J. Med. 24:337–352. 9. Pacht, E. R., P. Diaz, T. Clanton, J. Hart, and J. E. Gadek. 1997. Alveolar fluid glutathione decreases in asymptomatic HIV-seropositive subjects over time. Chest 112:785–788. 10. Cameron, P. U., S. A. Mallal, M. A. H. French, and R. L. Dawkins. 1990. Major histocompatibility complex genes influence the outcome of HIV-infection—ancestral haplotypes with C4 null alleles explain diverse HLA associations. Hum. Immunol. 29:282–295. 11. Hentges, F., A. Hoffmann, F. O. Dearaujo, and R. Hemmer. 1992. Prolonged clinically asymptomatic evolution after HIV-1 infection is marked by the absence of complement-C4 null alleles at the MHC. Clin. Exp. Immunol. 88:237–242. 12. Garred, P., H. O. Madsen, U. Balslev, B. Hofmann, C. Pedersen, J. Gerstoft, and A. Svejgaard. 1997. Susceptibility to HIV infection and progression of AIDS in relation to variant alleles of mannose-binding lectin. Lancet 349:236–240. 13. Khoo, S. H., L. Pepper, N. Snowden, A. H. Hajeer, P. Vallely, E. G. L. Wilkins, B. K. Mandal, and W. E. R. Ollier. 1997. Tumour necrosis factor c2 microsatellite allele is associated with the rate of HIV disease progression. AIDS 11:423–428. 14. Brinkman, B. M. N., I. P. M. Keet, F. Miedema, C. L. Verweij, and M. R. Klein. 1997. Polymorphisms within the human tumor necrosis factoralpha promoter region in human immunodeficiency virus type 1-seropositive persons. J. Infect. Dis. 175:188–190. 15. VanRij, R. P., S. Broersen, J. Goudsmit, R. A. Coutinho, and H. Schuitemaker. 1998. The role of a stromal cell-derived factor-1 chemokine gene variant in the clinical course of HIV-1 infection. AIDS 12:F85–F90. 16. Michael, N. L., G. Chang, L. G. Louie, J. R. Mascola, D. Dondero, D. L. Birx, and H. W. Sheppard. 1997. The role of viral phenotype and CCR-5 gene defects in HIV-1 transmission and disease progression. Nat. Med. 3:338–340. 17. Kwong, P. D., R. Wyatt, J. Robinson, R. W. Sweet, J. Sodroski, and W. A. Hendrickson. 1998. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393:648–659. 18. Alkhatib, G., S. S. Ahuja, D. Light, S. Mummidi, E. A. Berger, and S. K.
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