A number of human mAbs to HIVgp120 and transmembrane gp41 (2G12, b12, 2F5 and 4E10) show broad spectrum neutralizing properties [18–21,30–34]. They have been used successfully in passive immunization in macaques, and the complementary effect of two or three mAbs on HIV/SIV chimeric viruses was more effective than single mAb in preventing vaginal [22–25] or neonatal transmission [26–29]. Whether active preventive immunization will elicit high titres of such neutralizing antibodies is uncertain , and some may elicit cardiolipin autoreactivity .
An alternative strategy presented in this study addresses four molecules found in HIV-1 virions, which are unlikely to be conformationally dependent or hidden from the immune system. Antibodies to HLA-I or HLA-II are commonly found in multiparous women resulting from paternal foetal semiallogeneic antigens . Antibodies to HSP70 have been described in normal human participants , probably as a result of dendritic cells taking up HSP70 from T and B cells, especially under conditions of stress , but they have not been tested for HIV-1 inhibition. Antibodies to HIVgp120 envelope can be readily elicited, but usually they do not show broadly neutralizing antibodies. However, a few rare sera have been described from HIV-1-infected individuals who develop broadly neutralizing activity [40–46]. The data presented in this study raise the possibility that rare broadly neutralizing antibodies might be accounted for by a combination of HIVgp120, HLA and HSP70 antibodies.
As the antibodies target HLA-I, HLA-II, HIVgp120 and HSP70 molecules, each of these may affect HIV-1 entry and replication. HIVgp120 antibodies can neutralize HIV-1 and passive transfer of HLA-II antibodies protected macaques challenged with SIV [8,9]. HLA-II molecules promote assembly and budding of HIV-1 at the plasma membrane, and antibodies to these HLA-II molecules may affect HIV-1 replication . Immunization with HLA-I can also protect macaques from SIV infection . HSP70 plays an essential role in virion assembly and uncoating , HIV-1 preintegration complex formation , cell cycle arrest and apoptosis induced by HIV-1 viral protein R (VPR) . Furthermore, ATPase activity of HSP70 is required to maintain HIV-1 virion integrity .
In this study, we show systematically an increase in dose-dependent inhibition of clade B HIV-1 by single, double or triple combinations of antibodies to HLA-I or HLA-II with HSP70 and HIV-1 gp120. The lowest IC50 with clade B HIV-1 of 3.8 (±0.6) μg/ml and maximum inhibition of 96.7 (±0.9)% resulted to a greater extent from a combination of the three antibodies with HLA-II + HIVgp120 + HSP70 and to a lesser extent from the combination with HLA-I + HIVgp120 + HSP70 (4.7 ± 0.4 μg/ml and 92.8 ± 3.4%, respectively). Single or double antibody combination showed significantly higher IC50, from 6.5 to 19.0 μg/ml, and lower levels of HIV-1 inhibition. However, the decrease in IC50 was not synergistic or additive and the mechanism of the complementary neutralization activity needs to be elucidated. The proximity of the three molecules within the HIV-1 virion is not known, but it is likely that steric hindrance and conformational changes induced by the antibodies may favour neutralization. These results were not HLA-I or HLA-II allele specific, as comparable IC50 and dose-dependent inhibition was found with HLA-B27, B17, DR1, DR3 and DR4 antibodies. Thus, a combination of polyclonal human IgG antibodies with three diverse molecules expressed by HIV-1 virions are more effective, and in lower concentrations, than the corresponding single or double antibodies in inhibiting HIV-1 replication in human CD4 T cells.
To exclude other serum inhibitory factors and unrelated antibodies, mAb to the four molecules were studied. The results with the IgG mAb were consistent with those of polyclonal human IgG antibodies in that a progressive increase in dose-dependent inhibition was found with clade B HIV-1 by single, double or triple combination of mAb to HLA-I or HLA-II with HSP70 and HIV-1 gp120. The lowest IC50 of 3.5 (±1.3) μg/ml and maximum inhibition of 99 (±0.5)% resulted from a combination of the three mAbs to HLA-II + HIVgp120 + HSP70 and to a lesser extent from a combination of the mAbs to HLA-I + HIVgp120 + HSP70. Furthermore, a comparison of the broadly acting mAb b12 with the triple serum antibodies in the same assay showed an IC50 of 1.75 μg/ml with the mAb and 2.25 μg/ml with the antisera. Thus, the IC50 of the triple combination of antibodies compared favourably with the mAb b12, which is one of the ‘gold standards’ and has been very difficult to attain by conventional immunization.
To establish whether the combination of three polyclonal human antibodies elicit broadly neutralizing activity, further dose-dependent studies were pursued with two subtypes of HIV-1 clades A, C or D. These studies demonstrated that a combination of three human polyclonal antibodies to HLA-II with HIVgp120 and HSP70 elicited IC50 of 2.5–6.1 μg/ml and maximum inhibition of 75–100%, with the exception of clade A strains (92/RW/024) and clade C strain (93/IN/101). A direct comparison of the IC50 between antibodies to HIVgp120 and the triple antibody combination suggests that lower IC50 was achieved with the triple combination of antisera than the single gp120 antiserum with clades B, C and D (one strain each) but not with the two clade A strains and one clade C strain. These results should, however, be viewed in the context that the antisera to HLA and HSP70 were not from actively immunized persons, unlike the antisera to HIVgp120 that were from HIV-1-infected persons. Thus, broadly neutralizing antibody activity can be achieved with the combination of three polyclonal human antisera against three of the four clades of HIV-1 examined.
It is of interest that the antibody combinations failed to neutralize significantly CXCR4-using HIV-1 (LAI and group O). This was not studied further, but we can speculate that the polyclonal antibodies were most likely directed against CCR5-using viruses. HSP70 binds the CCR5 coreceptor  and inhibits infection mostly of R5 viruses , so although HSP70 antibodies would bind HSP70 both in the X4-type and R5-type HIV-1, the inhibitory effect would be seen only with the CCR5-using virus. Another aspect of HSP70 antibodies is that they are commonly found in normal human sera [38,39], can be increased on immunization and do not cause adverse effects.
The results of this investigation suggest that the trimolecular HLA–HIVgp120–HSP70 complex may constitute a novel vaccine strategy against HIV-1 infection, targeting not only the viral envelope gp120 but also defined host molecules expressed by the virus particles. The three components of such a vaccine have distinct functional properties. Alloimmune stimulation may engage up to 10% of T cells , elicit antibodies to HLA-II, which may bind CD4 molecules on T cells, macrophages and dendritic cells, and induce an array of cytokines and chemokines, which greatly enhance immunogenicity. HLA-I antibodies however may inhibit the cytotoxic function of CD8 T cells. HSP70 exerts mucosal and systemic adjuvanticities by stimulating chemokines, cytokines and maturation of dendritic cells [54,55]. HSP70 may also inhibit HIV-1 transmission by binding the CCR5 receptors [52,56], stimulating CC chemokines, which block CCR5, and upregulating the innate anti-HIV-1 APOBEC3G . HIVgp120 may elicit specific cellular and antibody responses to HIV-1, aided by the two HIV-1 non-cognate agents. Using the three antigens found in HIV virions is more likely to yield a more effective neutralizing antibody level than is possible by single antigen. This novel trimolecular vaccination strategy, utilizing HIV-1-specific and non-cognate immune responses, is unlikely to be subject to viral escape and will be examined in vivo in macaques to ascertain whether challenge with SHIV can be prevented or contained.
This work was supported partly by the European Community, Grants Empro (LSHP-CT-2003-503558) and Europrise (LSHP-CT-2006-037611), and by the Bill & Melinda Gates Foundation. We thank Dr P. Anton for the HIV-1 gp120 antibodies from HIV positive men and Dr R. Shattock for the PM1 cell line and clade O-HIV.
Dr K. Babaahmady was responsible for the neutralization studies with HIV-1, Dr L. Bergmeier carried out the screening of human sera for antibodies to HSP70 and separation of IgG from the human antisera. Dr T. Lehner conceived the project, was responsible for quality control and analyses of the data and wrote the paper with the aid of Drs Babaahmady and Bergmeier.
1. Tremblay MJ, Fortin J-F, Cantin R. The acquisition of host-encoded proteins by nascent HIV-1. Immunol Today 1998; 19:346–351.
2. Arthur LA, Bess JW Jr, Sowder RC II, Benveniste RE, Mann DL, Chermann J-C, Henderson LE. Cellular proteins bound to immunodeficiency viruses: implications for pathogenesis and vaccines. Science 1992; 258:1935–1938.
3. Gurer C, Cimarelli A, Luban J. Specific incorporation of heat shock protein 70 family members into primate lentiviral virions. J Virol 2002; 76:4666–4670.
4. O'Connor DH, Allen TM, Vogel TU, Jing P, DeSouza IP, Dodds E, et al
. Acute phase cytotoxic T lymphocyte escape is a hallmark of simian immunodeficiency virus infection. Nat Med 2002; 8:493–499.
5. Barouch DH, Kunstman J, Kuroda MJ, Schmitz JE, Santra S, Peyerl FW, et al
. Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature 2002; 415:335–339.
6. Lehner T, Shearer GM, Hackett CJ, Schultz A, Sharma OK. Workshop Summary. Alloimmunization as a strategy for vaccine design against HIV/AIDS. AIDS Res Hum Retroviruses 2000; 16:309–313.
7. Stott EJ. Anticell antibody in macaques. Nature 1991; 353:393.
8. Arthur LO, Bess JW Jr, Urban RG, Strominger JJ, Morton WR, Mann DL, et al
. Macaques immunized with HLA-DR are protected from challenge with simian immunodeficiency virus. J Virol 1995; 69:3117–3124.
9. Stott JE, Almond N, West W, Kent K, Cranage MP, Rudd E. Protection against simian immunodeficiency virus infection of macaques by cellular or viral antigens. Neuvieme Colloque Des Cent Gardes
10. Chan WL, Rodgers A, Grief C, Almond N, Ellis S, Flanagan B, et al
. Immunization with class I human histocompatibility leukocyte antigen can protect macaques against challenge infection with SIVmac-32H. AIDS 1995; 9:223–228.
11. Polyanskaya N, Sharpe S, Cook N, Leech S, Banks J, Dennis M, et al
. Antimajor histocompatibility complex antibody responses to simian B cells do not protect macaques against SIVmac infection. AIDS Res Hum Retroviruses 1997; 13:923–931.
12. Pinto LA, Sharpe S, Cohen DI, Shearer G. Alloantigen-stimulated anti-HIV activity. Blood 1998; 92:3346–3354.
13. Wang Y, Tao L, Mitchell E, Bravery C, Berlingieri P, Armstrong P, et al
. Allo-immunization elicits CD8+ T cell-derived chemokines, HIV suppressor factors and resistance to HIV infection in women. Nat Med 1999; 5:1004–1009.
14. Wang Y, Underwood J, Vaughan R, Harmer A, Doyle C, Lehner T. Allo-immunization elicits CCR5 antibodies, SDF-1 chemokines, and CD8-suppressor factors that inhibit transmission of R5 and X4 HIV-1 in women. Clin Exp Immunol 1999; 129:493–501.
15. Peters B, Whittall T, Babaahmady K, Gray K, Vaughan R, Lehner T. Effect of heterosexual intercourse on mucosal alloimmunisation and resistance to HIV-1 infection. Lancet 2004; 363:518–524.
16. Agnew LL, Kelly M, Howard J, Jeganathan S, Batterham M, French MA, et al
. Altered lymphocyte heat shock protein 70 expression in patients with HIV disease. AIDS 2003; 17:1985–1988.
17. Babahmady K, Oehlmann W, Singh M, Lehner T. Inhibition of HIV-1 infection of human CD4+ T cells by microbial HSP70 and the peptide epitope 407–426. J Virol 2007; 81:3354–3360.
18. Burton DR, Stanfield RL, Wilson IA. Antibody vs. HIV in a clash of evolutionary titans. Proc Natl Acad Sci U S A 2005; 102:14943–14948.
19. Scanlan CN, Pantophlet R, Wormald MR, Saphire EO, Stanfield R, Wilson IA, et al
. The broadly neutralizing antihuman immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of alpha 1–2 mannose residues on the outer face of gp120. J Virol 2002; 76:306–321.
20. Stiegler G, Kunert R, Purtscher M, Wolbank S, Voglauer R, Steindl F, Katinger H. A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses 2001; 17:1757–1765.
21. Zwick MB, Labrijn AF, Wang M, Spenlehauer C, Saphire EO, Binley JM. Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J Virol 2001; 75:10892–10905.
22. Mascola JR, Lewis MG, Stiegler G, Harris D, VanCott TC, Hayes D, et al
. Protection of macaques against pathogenic simian/human immunodeficiency virus 89.6P by passive transfer of neutralizing antibodies. J Virol 1999; 73:4009–4018.
23. Mascola JR, Stiegler G, VanCott TC, Katinger H, Carpenter CB, Hanson CE, et al
. Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Med 2000; 6:207–210.
24. Parren PW, Marx PA, Hessell AI, Luckay AJ, Harouse A, Cheng-Mayer J, et al
. Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro. J Virol 2001; 75:8340–8347.
25. Veazey RS, Shattock RJ, Pope MJ, Kirijan C, Jones J, Hu Q, et al
. Prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to HIV-1 gp120. Nat Med 2003; 9:343–346.
26. Baba T, Liska V, Hoffmann-Lehmann R, Vlasak J, Xu W, Ayehunie S, et al
. Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat Med 2000; 6:200–206.
27. Ferrantelli F, Hoffmann-Lehmann R, Rasmussen RA. Postexposure prophylaxis with human monoclonal antibodies prevented SHIV89.6P infection or disease in neonatal macaques. AIDS 2003; 17:301–309.
28. Hoffmann-Lehmann R, Vlasak J, Rasmussen RA, Smith BA, Baba TW, Liska V, et al
. Postnatal passive immunization of neonatal macaques with a triple combination of human monoclonal antibodies against oral simian-human immunodeficiency virus challenge. J Virol 2001; 75:7470–7480.
29. Hoffmann-Lehmann R, Vlasak I, Rasmussen RA, Jiang S, Li PL, Baba TW, et al
. Postnatal pre and postexposure passive immunization strategies: protection of neonatal macaques against oral simian-human immunodeficiency virus challenge. J Med Primatol 2002; 31:109–119.
30. Muster T, Steindl F, Purtscher M, Hayes D, Louder MK, Brown CR, et al
. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type I. J Virol 1993; 67:6642–6647.
31. Saphire EO, Parren PW, Pantophlet R, Zwick MB, Morris GM, Rudd PM, et al
. Crystal structure of a neutraslizing human IgG against HIV-1: a template for vaccine design. Science 2001; 293:1155–1159.
32. Wyatt R, Sodroski J. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 1998; 280:1884–1888.
33. Zwick MB, Parren PW, Saphire EO, Church S, Wang M, Scott JK, et al
. Molecular features of the broadly neutralizing immunoglobulin G1 b12 required for recognition of human immunodeficiency virus type 1 gp120. J Virol 2003; 77:5863–5876.
34. Kwong PD, Wyatt R, Majeed S, Robinson J, Sweet RW, Sodroski J, Hendrickson WA. Structures of HIV-1 gp120 envelope glycoproteins from laboratory-adapted and primary isolates. Structure 2000; 8:1329–1339.
35. Montefiori DC. Neutralizing antibodies take a swipe at HIV in vivo
. Nat Med 2005; 11:593–594.
36. Haynes BF, Fleming J, St-Clair EW, Katinger H, Stiegler G, Kunert R, et al
. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 2005; 308:1906–1908.
37. Morin-Papunen L, Tiilikainen A, Hartikainen-Sorri A-L. Maternal HLA immunization during pregnancy: presence of anti HLA antibodies in half of multigravidous women. Med Microbiol 1984; 62:323–325.
38. Pockley AG, Shepherd J, Corton JM. Detection of heat shock protein 70 (hsp70) and anti-Hsp70 antibodies in the serum of normal individuals. Immunol Invest 1998; 27:367–377.
39. Hunter-Lavin C, Davies EL, Bacelar MMFVG, Marshall MJ, Andrew SM, Williams JHH. Hsp70 release from peripheral blood mononuclear cells. Biomed Biophys Res Commun 2004; 324:511–517.
40. Dhillon AK, Donners H, Pantophlet R, Johnson WE, Decker JM, Shaw GM, et al
. Dissecting the neutralizing antibody specificities of broadly neutralizing sera from human immunodeficiency virus type 1 infected donors. J Virol 2007; 81:6548–6562.
41. Haynes BF, Montefiori DC. Aiming to induce broadly reactive neutralizing antibody responses with HIV-1 vaccine candidates. Expert Rev Vaccines 2006; 5:579–595.
42. Li X, Migueles SA, Welcher B, Svehla K, Phogat A, Louder MK, et al
. Broad HIV-1 neutralization mediated by CD4-binding site antibodies. Nat Med 2007; 13:1032–1034.
43. Stamatos NM, Mascola JR, Kalyanaraman VS, Louder MK, Frampton LM, Birx DL, VanCott TC. Neutralizing antibodies from the sera of human immunodeficiency virus type 1 infected individuals bind to monomeric gp120 and oligomeric gp140. J Virol 1998; 72:9656–9667.
44. Beirnaert E, De Zutter S, Janssens W, van der Groen G. Potent broad cross-neutralizing sera inhibit attachment of primary HIV-1 isolates (groups M and O) to peripheral blood mononuclear cells. Virology 2001; 281:305–314.
45. Cham F, Zhang PF, Heyndrickx L, Bouma P, Zhong P, Katinger H, et al
. Neutralization and infectivity characteristics of envelope glycoproteins from human immunodeficiency virus type 1 infected donors whose sera exhibit broadly cross-reactive neutralizing activity. Virology 2006; 347:36–51.
46. Quinnan GV Jr, Zhang PF, Fu DW, Dong M, Alter HJ. Expression and characterization of HIV type 1 envelope protein associated with a broadly reactive neutralizing antibody response. AIDS Res Hum Retrovir 1999; 15:561–570.
47. Finzi A, Orthwein A, Mercier J, Cohen EA. Productive human immunodeficiency virus type 1 assembly takes place at the plasma membrane. J Virol 2007; 81:7476–7490.
48. Chappell TG, Welch WJ, Schlossman DM, Palter KB, Schlesinger MJ, Rothman JE. Uncoating ATPase is a member of the 70 kilodalton family of stress proteins. Cell 1996; 45:3–13.
49. Agostini I, Popov LS, Li J, Dubrovsky L, Hao T, Bukrinsky M. Heat-shock protein 70 can replace viral protein R of HOV-1 during nuclear import of the viral preintegration complex. Exp Eye Res 2000; 259:398–403.
50. Lordanskiy S, Zhao Y, Dubrovsky L, Lordanskaya T, Chen M, Liang D, Bukrinsky M. Heat shock protein 70 protects cells from cell cycle arrest and apoptosis induced by human immunodeficiency virus type 1 viral protein R. J Virol 2004; 78:9697–9704.
51. Gurer C, Hoglund A, Hoglund S, Luban J. ATPγS disrupts human immunodeficiency virus type 1 virion core integrity. J Virol 2005; 79:5557–5567.
52. Whittall T, Wang Y, Younson J, Kelly C, Bergmeier LA, Peters B, et al
. Interaction between the CCR5 chemokine receptors and microbial HSP70. Eur J Immunol 2006; 36:2304–2314.
53. Blattman JN, Antia R, Sourdive DJD, Wang X, Kaech SM, Murali-Krishna K, et al
. Estimating the precursor frequency of naïve antigen-specific CD8 T cells. J Exp Med 2002; 195:657–664.
54. Lehner T, Wang Y, Whittall T, McGowan E, Kelly CG, Singh M. Functional domains of HSP70 stimulate generation of cytokines and chemokines, maturation of dendritic cells and adjuvanticity. Biochem Soc Trans 2004; 32:629–632.
55. Wang Y, Kelly CG, Singh M, McGowan EG, Carrara A-S, Bergmeier LA, Lehner T. Stimulation of Th1-polarizing cytokines, C-C chemokines, maturation of dendritic cells and adjuvant function by the peptide binding fragment of heat shock protein 70. J Immunol 2002; 169:2422–2429.
56. Floto RA, MacAry PA, Boname JM, Mien TS, Kampmann B, Hair JR, et al
. Dendritic cell stimulation by mycobacterial Hsp70 is mediated through CCR5. Science 2006; 314:454–458.
57. Pido-Lopez J, Whittall T, Wang Y, Bergmeier LA, Babaahmady K, Singh M, Lehner T. Stimulation of cell surface CCR5 and CD40 molecules by their ligands or by HSP70 upregulates APOBEC3G expression in CD4+
T cells and dendritic cells. J Immunol 2007; 178:1671–1679.