Evolution of Live-Attenuated HIV Vaccines

ABSTRACT

Despite intensive research since the viral pathogen was discovered some 25 years ago, not much progress has been reported on the development of a safe vaccine that protects against human immunodeficiency virus type 1. A vaccine approach that has been abandoned because its safety cannot be guaranteed is the single vaccine candidate that provides good protection in the macaque model, a live-attenuated variant of the simian immunodeficiency virus. The attenuated virus will cause a low-grade, but persistent infection that allows optimization of viral replication kinetics over time by spontaneous virus evolution, which may increase viral pathogenicity. In this article, we discuss innovative strategies to overcome this hurdle, including the generation of "single-cycle" viruses and a conditionally replicating HIV-1 variant.

Vaccines that consist of a live-attenuated virus strain have proven to be very successful at inducing protective immunity against pathogenic viruses such as those causing smallpox, polio, and measles.¹ Research on the development of a live-attenuated human immunodeficiency virus (HIV) vaccine has predominantly been performed in macaques that are infected with pathogenic simian immunodeficiency virus (SIV). Attenuation occurs by the deletion of several accessory functions from the viral genome, either individually or in combination.^2–5 The majority of monkeys vaccinated with such deletion mutants of SIV can efficiently control replication of pathogenic challenge virus strains. However, the attenuated virus could revert to virulence and cause disease over time in vaccinated animals, especially in neonates.^6–10 Similarly, some of the long-term nonprogressors of the Sydney Blood Bank Cohort, infected with a naturally attenuated HIV-1 variant with deletions in the nef and long terminal repeat (LTR) sequences eventually progressed to acquired immunodeficiency syndrome (AIDS).¹¹ An HIV-1 Δ3 variant with deletions in the vpr, nef, and LTR sequences regained substantial replication capacity in long-term cell culture infections by acquiring compensatory changes in the viral genome.¹² These results highlight the genetic instability and evolutionary capacity of attenuated SIV/HIV strains, which pose a serious safety risk for any future experimentation with live-attenuated HIV vaccines in humans. Novel strategies are needed to reconsider this vaccination approach because many other vaccine attempts have thus far failed to provide protection.¹³

(CHRISTINE BALDERAS, GETTY IMAGES)

The Safety Issue of Live-Attenuated HIV-1 Variants

The major safety problem of live-attenuated HIV/SIV vaccine strains is related to the persistent replication and consequent evolution of the attenuated virus. In combination with the error-prone replication machinery of the virus, this ongoing low-level replication may eventually lead to the appearance of fitter and more pathogenic virus variants. To improve safety, the vaccine strain can be further attenuated through additional deletions or mutations in accessory genes or regulatory elements. This further reduces the pathogenic properties of the virus, but at the same time also reduces the vaccine efficacy.^14–15 As an alternative strategy to prevent evolution toward a pathogenic variant, replication of the vaccine virus should be limited to the extent and time window that is required to provide full protection. For instance, virus replication can be stopped a few weeks after vaccination by administrating antiviral drugs.¹⁶ Although this is a good research strategy for macaque studies to address whether ongoing replication of the vaccine strain is needed for protection, application in humans seems problematic because long-term virus inhibition will require continuous drug administration, and the virus may develop drug resistance. Alternatively, a virus that can execute only a single round of replication can be used as a vaccine.^17–20 However, because of the limited replication, such a single-cycle virus vaccine may be less potent for inducing protective immunity. We and others previously presented an alternative approach that uses a conditionally live HIV or SIV variant.^21–25 We will discuss some of these approaches in this article.

Single-Cycle Virus Variants

To reduce the risks associated with ongoing replication and evolution of live-attenuated SIV strains, single-cycle SIV variants (scSIV) have been designed. The initial scSIV variant was designed to use an artificial tRNA primer for reverse transcription that was exclusively present in the modified producer cell line, but not in other cells.²⁰ In addition, attenuating deletions were introduced in the vif, vpr, vpx, and nef genes. After a single intravenous injection in rhesus macaques, peak viral RNA levels of 10³ to 10⁴ copies/mL plasma were observed, indicating efficient expression of scSIV in the vaccinee. However, the vaccine doses used could not protect macaques from subsequent intravenous challenges with the pathogenic wild-type virus. A second approach for producing scSIV was based on Gag-Pol complementation of an SIV genome that is deficient for Pol expression as a result of a combination of mutations in the frameshift site that controls Pol translation.¹⁷ Four macaques were inoculated intravenously with three concentrated doses of scSIV, and viral loads peaked between 10⁴ and 10⁵ RNA copies/mL.¹⁸ On challenge, all animals became infected, but two of these animals were able to contain their viral loads below 2,000 RNA copies/mL as late as 35 weeks into the chronic phase of infection. These observations are encouraging and endorse future studies aimed at improving the protection.

Conditional Replicating Virus Variants

To improve the safety of a live-attenuated HIV-SIV vaccine strain, one must be able to shut-off virus replication once the vaccine has done its job. We introduced a genetic switch in the HIV-1 genome to control its replication. HIV gene expression and replication are naturally controlled by the viral Tat protein that binds to the 5' trans-acting responsive (TAR) region in the nascent RNA transcript to enhance transcription.²⁶ For constructing a conditionally live HIV variant, this Tat–TAR regulatory mechanism was inactivated by mutation and functionally replaced by components of the doxycycline(dox)-inducible gene expression system (Tet-On system).²⁷ This E.coli-derived gene-expression system is controlled by the rtTA protein, and binding of dox triggers a conformational switch that allows binding this protein to tet operator (tetO) elements and activation of transcription from the downstream positioned promoter. Thus, we introduced tetO elements in the LTR promoter and the rtTA gene in place of the nef gene. Transcription of this HIV-rtTA construct is activated by binding the dox-rtTA complex to the tetO-LTR promoter, and this virus replicates exclusively when dox is administered. After vaccination with this virus, replication can be temporarily activated by transient dox administration to the extent needed for induction of protective responses. The initial HIV-rtTA construct has been improved significantly by spontaneous virus evolution in prolonged cell culture infections.^28–34 We have shown efficient and dox-dependent virus replication not only in vitro in T cell lines, but also ex vivo in human lymphoid tissue.³⁵ An equivalent SIVmac variant was recently constructed that yielded promising results in vaccination tests in rhesus macaques (unpublished results).

Is Double Control Needed?

We realize that safety remains a major concern for drug-controlled virus variants. In fact, we constructed an HIV-1 variant that depends not only on dox for gene expression, but also on the T20 peptide for cell entry.³⁶ T20 (Fuzeon) is a 36-mer peptide that mimics part of the HR2 domain of the envelope protein that is intrinsically involved in entry of the virus into the cell.³⁷ We described the evolution of a T20-dependent HIV-1 variant in a patient on T20 therapy.³⁸ This virus acquired two substitutions that created a hyperactive envelope protein. Further analysis revealed that the T20 peptide can rescue this hyperfusogenic protein by preventing the premature conformational switch, thus restoring virus infectivity and replication.³⁹ Introducing these two mutations in HIV-rtTA resulted in a virus that replicates exclusively in the combined presence of dox and T20.³⁶ Subsequent withdrawal of these inducers efficiently blocks viral replication and prevents ongoing evolution.

Dox-Controlled Virus for Virotherapy

We explored the possibility to use viruses based on HIV-1 strains that use CD4 and CXCR4 for cell entry as a therapeutic virus against malignancies such as T-lymphoblastic leukemia/lymphoma, NK leukemia, and some myeloid leukemias.⁴⁰ The dox-controllable HIV-rtTA approach was combined with the design of a minimized HIV-1 variant for developing a virotherapy for cancer.²¹ This mini-HIV-rtTA variant lacks several nonessential genes and has lost the ability to replicate in normal primary cells, but this variant is still able to replicate in leukemic T-cell lines.⁴¹ This virus can efficiently and exclusively remove leukemic cells from a mixed culture with untransformed cells. In a therapeutic setting, the minimized virus can be used to target leukemic cells in the presence of dox. This will result in a self-limiting viral infection because the target cells are killed by the virus. Withdrawing dox provides an additional safety feature to block ongoing replication after the leukemic cells are removed.

Dox-Controlled Virus for Delivering RNAi Therapeutics

We also explored the potential of the HIV-rtTA variant as a replicating vector for the efficient delivery of inhibitory gene cassettes that are based on the RNA interference (RNAi) mechanism. More specifically, we introduced an RNA polymerase III-driven short hairpin RNA (shRNA) cassette against wild-type HIV-1 sequences in the context of the dox-dependent virus.⁴² The shRNA targets the viral nef sequence, which is present in wild-type HIV-1 but not in the HIV-rtTA vector where the nef gene has been replaced by the rtTA gene. A spreading infection of this therapeutic HIV-rtTA-shRNAnef variant in HIV-susceptible cells can be controlled by transient dox treatment. Subsequent dox withdrawal generates cells that contain a silent integrated provirus with a constitutively active shRNAnef expression cassette. As a result, cells are harnessed with shRNAs that efficiently inhibit replication of wild-type HIV-1. This strategy seems particularly suitable for patients infected with a multidrug-resistant virus that can no longer be treated with the current antivirals. This HIV-rtTA-shRNAnef variant may allow interesting combinations of vaccination and RNAi-inhibition strategies. When used as a prophylactic vaccine, the RNAi cargo of this virus will protect all infected cells against a future exposure to HIV-1, thus boosting vaccine protection. When used as a therapeutic virus, the vaccine effect may boost the RNAi-mediated virus inhibition.

Conclusion

Obvious safety concerns remain for developing replicating vectors based on the human pathogen HIV-1. One of the major concerns is that attenuated HIV-1 variants also cause a chronic infection. This fact, combined with the high mutation and recombination rate of HIV-1, may result in the generation of variants with altered replication characteristics over time. However, the dox-controlled HIV-rtTA variant will cause a latent infection on dox-withdrawal, with silent integrated proviruses that will less likely contribute to ongoing virus evolution because they are transcriptionally inactive. Another concern is that the vector may integrate near the 5' end of a proto-oncogene. In this position, the viral LTR-promoter may activate proto-oncogene expression, which could result in cell proliferation, and ultimately cause cancer. Such insertional oncogenesis occurred in 5 out of 20 patients who were treated with a gamma-retroviral vector,^43,44 but the new generation lentiviral vectors were designed to be more safe, which is upheld in recent trials.^45,46 The tetO-LTR promoter in our HIV-rtTA vectors is inactive on dox-withdrawal, which will strongly reduce the risk of activation of adjacent genes.

We recently constructed a similar dox-dependent SIV variant, which is currently being used to study the efficacy and safety of a conditionally live virus vaccine against AIDS in macaques.⁴⁷ This SIV variant may be a particularly attractive tool to study the correlates of immune protection on vaccination because the level and duration of replication can be controlled by dox administration. As a next step, the genetic stability and immunogenicity of the HIV-rtTA variant could be tested in mice with a humanized immune system.^48,49 These results should indicate whether we can proceed on the risky path toward a live-attenuated HIV-1 vaccine.

Acknowledgements

This research was funded by the Dutch AIDS Foundation (Aids Fonds Netherlands grants 7007, 2005–022, 2007–025), the Technology Foundation STW (applied science division of NWO and the technology program of the Ministry of Economic Affairs, The Netherlands), Zon-Medical Sciences (MW; VICI grant), and NWO-Chemical Sciences (CW; TOP grant).

BEN BERKHOUT, PhD, is head of the Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands, 31.20 5663396, b.berkhout@amc.uva.nl

References

1. Lauring AS, Jones JO, Andino R. Rationalizing the development of live attenuated virus vaccines. Nat Biotechnol. 2010;28(6):573–9.

2. Johnson RP, Desrosiers RC. Protective immunity induced by live attenuated simian immunodeficiency virus. Curr Opin Immunol. 1998;10(4):436–43.

3. Mills J, Desrosiers R, Rud E, Almond N. Live attenuated HIV vaccines: proposal for further research and development. AIDS Res. Hum Retroviruses 2000;16:1453–61.

4. Whitney JB, Ruprecht RM. Live attenuated HIV vaccines: pitfalls and prospects. Curr Opin Infect Dis. 2004;17(1):17–26.

5. Koff WC, Johnson PR, Watkins DI, Burton DR, Lifson JD, Hasenkrug KJ, et al. HIV vaccine design: insights from live attenuated SIV vaccines. Nat Immunol. 2006;7(1):19–23.

6. Baba TW, Jeong YS, Penninck D, Bronson R, Greene MF, Ruprecht RM. Pathogenicity of live, attenuated SIV after mucosal infection of neonatal macaques. Science. 1995;267:1820–5.

7. Baba TW, Liska V, Khimani AH, Ray NB, Dailey PJ, Penninck D, et al. Live attenuated, multiply deleted simian immunodeficiency virus causes AIDS in infant and adult macaques. Nat Med. 1999;5:194–203.

8. Chakrabarti LA, Metzner KJ, Ivanovic T, Cheng H, Louis-Virelizier J, Connor RI, Cheng-Mayer C. A truncated form of Nef selected during pathogenic reversion of simian immunodeficiency virus SIVmac239Deltanef increases viral replication. J Virol. 2003;77(2):1245–56.

9. Whatmore AM, Cook N, Hall GA, Sharpe S, Rud EW, Cranage MP. Repair and evolution of nef in vivo modulates simian immunodeficiency virus virulence. J Virol. 1995;69:5117–23.

10. Hofmann-Lehmann R, Vlasak J, Williams AL, Chenine AL, McClure HM, Anderson DC, O'Neil S, Ruprecht RM. Live attenuated, nef-deleted SIV is pathogenic in most adult macaques after prolonged observation. AIDS. 2003;24:17(2):157–66.

11. Churchill MJ, Rhodes DI, Learmont JC, Sullivan JS, Wesselingh SL, Cooke IR, Deacon NJ, Gorry PR. Longitudinal analysis of human immunodeficiency virus type 1 nef/long terminal repeat sequences in a cohort of long-term survivors infected from a single source. J Virol. 2006;80(2):1047–52.

12. Berkhout B, Verhoef K, van Wamel JLB, Back B. Genetic instability of live-attenuated HIV-1 vaccine strains. J Virol. 1999;73:1138–45.

13. Berkhout B, Paxton WA. HIV vaccine: it may take two to tango, but no party time yet. Retrovirol. 2009;6:88.

14. Lohman BL, McChesney MB, Miller CJ, McGowan E, Joye SM, van Rompay KK, et al. A partially attenuated simian immunodeficiency virus induces host immunity that correlates with resistance to pathogenic virus challenge. J Virol. 1994;68:7021–9.

15. Wyand MS, Manson KH, Garcia-Moll M, Montefiori D, Desrosiers RC. Vaccine protection by a triple deletion mutant of simian immuodeficiency virus. J Virol. 1996;70:3724–33.

16. Lifson JD, Rossio JL, Piatak M Jr., Parks T, Li L, Kiser R, et al. Role of CD8(+) lymphocytes in control of simian immunodeficiency virus infection and resistance to rechallenge after transient early antiretroviral treatment. J Virol. 2001;75(21):10187–99.

17. Evans DT, Bricker JE, Desrosiers RC. A novel approach for producing lentiviruses that are limited to a single cycle of infection. J Virol. 2004;78(21):11715–25.

18. Evans DT, Bricker JE, Sanford HB, Lang S, Carville A, Richardson BA, et al. Immunization of macaques with single-cycle simian immunodeficiency virus (SIV) stimulates diverse virus-specific immune responses and reduces viral loads after challenge with SIVmac239. J Virol. 2005;79(12):7707–20.

19. Falkensammer B, Rubner B, Hiltgartner A, Wilflingseder D, Stahl HC, Kuate S, et al. Role of complement and antibodies in controlling infection with pathogenic simian immunodeficiency virus (SIV) in macaques vaccinated with replication-deficient viral vectors. Retrovirol. 2009;6:60.

20. Kuate S, Stahl-Hennig C, ten HP, Heeney J, Uberla K. Single-cycle immunodeficiency viruses provide strategies for uncoupling in vivo expression levels from viral replicative capacity and for mimicking live-attenuated SIV vaccines. Virol. 2003;313(2):653–62.

21. Verhoef K, Marzio G, Hillen W, Bujard H, Berkhout B. Strict control of human immunodeficiency virus type 1 replication by a genetic switch: Tet for Tat. J Virol. 2001;75(2):979–87.

22. Smith SM, Khoroshev M, Marx PA, Orenstein J, Jeang KT. Constitutively dead, conditionally live HIV-1 genomes. Ex vivo implications for a live virus vaccine. J Biol Chem. 2001;276(34):32184–90.

23. Berkhout B, Marzio G, Verhoef K. Control over HIV-1 replication by an antibiotic; a novel vaccination strategy with a drug-dependent virus. Virus Res. 2002;82(1-2):103–8.

24. Das AT, Zhou X, Vink M, Klaver B, Berkhout B. Conditional live virus as a novel approach towards a safe live attenuated HIV vaccine. Expert Rev Vaccines. 2002;1(3):293–301.

25. Das AT, Verhoef K, Berkhout B. A conditionally replicating virus as a novel approach toward an HIV vaccine. Methods Enzymol. 2004;388:359–79.

26. Berkhout B, Silverman RH, Jeang KT. Tat trans-activates the human immunodeficiency virus through a nascent RNA target. Cell 1989;59(2):273–82.

27. Baron U, Bujard H. Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods Enzymol. 2000;327:401–21.

28. Marzio G, Verhoef K, Vink M, Berkhout B. In vitro evolution of a highly replicating, doxycycline-dependent HIV for applications in vaccine studies. Proc Natl Acad Sci USA. 2001;98(11):6342–7.

29. Marzio G, Vink M, Verhoef K, de Ronde A, Berkhout B. Efficient human immunodeficiency virus replication requires a fine-tuned level of transcription. J Virol. 2002;76(6):3084–8.

30. Das AT, Zhou X, Vink M, Klaver B, Verhoef K, Marzio G, Berkhout B. Viral evolution as a tool to improve the tetracycline-regulated gene expression system. J Biol Chem. 2004;279(18):18776–82.

31. Zhou X, Vink M, Klaver B, Verhoef K, Marzio G, Das AT, Berkhout B. The genetic stability of a conditional-live HIV-1 variant can be improved by mutations in the Tet-On regulatory system that restrain evolution. J Biol Chem. 2006;281(25):17084–91.

32. Zhou X, Vink M, Klaver B, Berkhout B, Das AT. Optimization of the Tet-On system for regulated gene expression through viral evolution. Gene Ther. 2006;13(19):1382–90.

33. Zhou X, Vink M, Berkhout B, Das AT. Modification of the Tet-On regulatory system prevents the conditional-live HIV-1 variant from losing doxycycline-control. Retrovirol. 2006;3(1):82.

34. Das AT, Harwig A, Vrolijk MM, Berkhout B. The TAR hairpin of human immunodeficiency virus type-1 can be deleted when not required for Tat-mediated activation of transcription. J Virol. 2007;81(14):7742–8.

35. Kiselyeva Y, Ito Y, Lima RG, Grivel JC, Das AT, Berkhout B, Margolis LB. Depletion of CD4 T lymphocytes in human lymphoid tissue infected ex vivo with doxycycline-dependent HIV-1. Virol. 2004;10;328(1):1–6.

36. Das AT, Baldwin CE, Vink M, Berkhout B. Improving the safety of a conditional-live human immunodeficiency virus type 1 vaccine by controlling both gene expression and cell entry. J Virol. 2005;79(6):3855–8.

37. Baldwin CE, Sanders RW, Berkhout B. Inhibiting HIV-1 entry with fusion inhibitors. Curr Med Chem. 2003;10(17):1633–42.

38. Baldwin CE, Sanders RW, Deng Y, Jurriaans S, Lange JM, Lu M, Berkhout B. Emergence of a drug-dependent human immunodeficiency virus type 1 variant during therapy with the T20 fusion inhibitor. J Virol. 2004;78(22):12428–37.

39. Baldwin C, Berkhout B. Mechanistic studies on a T20-dependent HIV-1 variant. J Virol. 2008;82:7735–40.

40. Jeeninga RE, Jan B, Van der Linden B, Van den Berg H, Berkhout B. Construction of a minimal HIV-1 variant that selectively replicates in leukemic derived T-cell lines: towards a new virotherapy approach. Cancer Res. 2005;65(8):3347–55.

41. Jeeninga RE, Jan B, Van den Berg H, Berkhout B. Construction of doxycyline-dependent mini-HIV-1 variants for the development of a virotherapy against leukemias. Retrovirol. 2006;3:64.

42. Westerhout EM, Vink M, Haasnoot PC, Das AT, Berkhout B. A conditionally replicating HIV-based vector that stably expresses an antiviral shRNA against HIV-1 replication. Mol Ther. 2006;14(2):268–75.

43. Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E, et al. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Invest. 2008;118(9):3132–42.

44. Howe SJ, Mansour MR, Schwarzwaelder K, Bartholomae C, Hubank M, Kempski H, Brugman MH, Pike-Overzet K, Chatters SJ, de RD, et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest. 2008;118(9):3143–50.

45. Levine BL, Humeau LM, Boyer J, MacGregor RR, Rebello T, Lu X, et al. Gene transfer in humans using a conditionally replicating lentiviral vector. Proc Natl Acad Sci USA. 2006;103(46):17372–7.

46. Cartier N, Hacein-Bey-Abina S, Bartholomae CC, Veres G, Schmidt M, Kutschera I, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science. 2009;326(5954):818–23.

47. Das AT, Klaver B, Harwig A, Vink M, Ooms M, Centlivre M, Berkhout B. Construction of a doxycycline-dependent simian immunodeficiency virus reveals a non-transcriptional function of Tat in viral replication. J Virol. 2007;81(20):11159–69.

48. An DS, Poon B, Ho Tsong FR, Weijer K, Blom B, Spits H, Chen IS, Uittenbogaart CH. Use of a novel chimeric mouse model with a functionally active human immune system to study human immunodeficiency virus type 1 infection. Clin Vaccine Immunol. 2007;14(4):391–6.

49. Ter Brake O, Legrand N, von Eije KJ, Centlivre M, Spits H, Weijer K, Blom B, Berkhout B. Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)(c)(-/-)) mouse model. Gene Ther. 2009;16(1):148–53.