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Safety concerns remain for developing replicating vectors based on the pathogen human immunodeficiency virus type 1.
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 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.
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.
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).
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.
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.
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.
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.
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, email@example.com
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