Generation and Characterization of a Defective HIV-1 Virus as an Immunogen for a Therapeutic Vaccine

Abstract

BackgroundThe generation of new immunogens able to elicit strong specific immune responses remains a major challenge in the attempts to obtain a prophylactic or therapeutic vaccine against HIV/AIDS. We designed and constructed a defective recombinant virus based on the HIV-1 genome generating infective but non-replicative virions able to elicit broad and strong cellular immune responses in HIV-1 seropositive individuals.ResultsViral particles were generated through transient transfection in producer cells (293-T) of a full length HIV-1 DNA carrying a deletion of 892 base pairs (bp) in the pol gene encompassing the sequence that codes for the reverse transcriptase (NL4-3/ΔRT clone). The viral particles generated were able to enter target cells, but due to the absence of reverse transcriptase no replication was detected. The immunogenic capacity of these particles was assessed by ELISPOT to determine γ-interferon production in a cohort of 69 chronic asymptomatic HIV-1 seropositive individuals. Surprisingly, defective particles produced from NL4-3/ΔRT triggered stronger cellular responses than wild-type HIV-1 viruses inactivated with Aldrithiol-2 (AT-2) and in a larger proportion of individuals (55% versus 23% seropositive individuals tested). Electron microscopy showed that NL4-3/ΔRT virions display immature morphology. Interestingly, wild-type viruses treated with Amprenavir (APV) to induce defective core maturation also induced stronger responses than the same viral particles generated in the absence of protease inhibitors.ConclusionsWe propose that immature HIV-1 virions generated from NL4-3/ΔRT viral clones may represent new prototypes of immunogens with a safer profile and stronger capacity to induce cellular immune responses than wild-type inactivated viral particles.

HIV-1 replicates in human osteoclasts and enhances their differentiation in vitro

“…Infectious units (IFU) of each virus stock were determined by p24 immuno-staining of infected MAGIC5 cells, susceptible to both R5 and X4 tropic HIV-1 [ 22 ]. Inactivated viruses were prepared by incubating the viral supernatants with 500 mM aldrithiol-2 (Sigma) at 4°C overnight [ 23 ]. For infection of macrophages and osteoclasts, cells were incubated for 6 hours with JR-FL or NL4-3, washed three times with OPBM, and further cultured for the indicated times.…”

Use of RT-Defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-Infected Individuals

“…As it is shown in figure 1A the proportion of patients with positive immune responses against NL4-3/ΔRT (ΔRT) virions (55%) was significantly higher than to wild type (25%) (p<0.001) [8] independently of the use of R5 or X4 receptors by viral envelopes (51–53%). The magnitude of the response (SFC/10 6 cells) elicited by both the X4 and R5 ΔRT variants was also higher than with wild type virus (p<0.001 and p<0.01, respectively), fact that confirmed that this difference was also applicable to the intensity of the response (fig.…”

“…1) in comparison with AT-2 inactivated wild type virions. The potential mechanisms responsible of such differences have been discussed elsewhere [8] but it seems that probably, the maturation status of our defective particle could imply a major stability which allows increased peptide presentation and processing by APC. This result is really interesting because the magnitude of a vaccine-induced responses may be related to the potency and frequency of immunization, which may also influence durability of the response [34].…”

“…The X4 HIV strains used were the NL4-3 and the NL4-3/ΔRT [8]. R5 HIV strains were the NL4-3/ΔRT/Δenv[BaL] and NL4-3/ΔRT/Δenv[AC10], obtained by modifications of the NL4-3/ΔRT (patent code EP 11382103.7).…”

“…In this sense and attending to this differential and important role for RT, our group has recently reported the construction and characterization of retrotranscriptase defective virions (NL4-3/ΔRT) [8] to improve safety as a future candidate for being used in HIV therapeutic vaccines. These attenuated constructs prevent viral integration and further replication thus avoiding evolution towards a pathogenic variant with a total deletion of the gene coding RT.…”