Therapeutic immunization of highly active antiretroviral therapy-treated HIV-1-infected patients: safety and immunogenicity of an HIV-1 gag/poly-epitope DNA vaccine

In view of the global emergency posed by lack of access to highly active antiretroviral therapy (HAART) and the limitations of current drug regimens, alternative therapeutic strategies are urgently needed. Cellular immune responses elicited by HIV-1 exert some control over virus replication, therefore the enhancement of HIV-1-specific responses by therapeutic vaccination might lead to viral containment without HAART. We evaluated the safety and immunogenicity, in HIV-1-infected individuals under HAART suppression, of a DNA vaccine, pTHr.HIVA.

The candidate vaccine, pTHr.HIVA, encodes an immunogen, HIVA, comprising HIV-1 clade A p24/p17 fused to a string of cytotoxic T-cell epitopes, and has been described in detail elsewhere ^[1]. Approvals were obtained from the local Research Ethics Committee and the United Kingdom Department of Health Gene Therapy Advisory Committee to evaluate the safety and immunogenicity of this vaccine in a cohort of HIV-1-seropositive individuals who had received continuous highly active antiretroviral therapy (HAART) for at least 12 months (median 40, range 13-60 months). Ten individuals (eight men, two women; median age 39 years), were recruited from two outpatient clinics in Oxford. All subjects gave written informed consent and had had sustained undetectable viraemia (< 400 copies/ml, Roche Amplicor assay; Roche Diagnostic Systems, UK) and CD4 T-cell counts greater than 300 cells/μl (median 550 cells/μl) for at least 6 months. They remained on the same drug regimen throughout the study period. Study participants received two intramuscular injections of 500 μg pTHr.HIVA (produced according to good manufacturing practice) on days 0 and 21 into the deltoid region of alternate arms. Safety monitoring consisted of a physical examination, observation of vital signs and the measurement of laboratory parameters including the CD4 T lymphocyte count, plasma viral load and anti-DNA antibodies, and was at each timepoint indicated (Fig. 1). Subjects were observed in the clinic after each immunization to assess reactogenicity and were asked to complete a symptom diary. The vaccination site was reviewed at follow-up at 2 weeks.

Fig. 1

Immunogenicity was assessed using a previously validated IFN-γ enzyme-linked immunospot (ELISPOT) assay ^[2] to quantify ex-vivo peptide-stimulated CD8 and CD4 T-cell responses simultaneously. Synthetic high-pressure liquid chromatography-purified peptides (Sigma-Genosys, Cambridge, UK) were used in pools of 22-23 peptides at a final concentration of 4 μg/ml. 'HIVA' pools 1-4 comprised peptides based on the p24/p17 sequence of the HIVA immunogen (15-mers overlapping by 11 amino acids). Positive control peptides comprised known CD8 T-cell epitopes derived from cytomegalovirus, Epstein-Barr virus and influenza virus proteins. Freshly isolated peripheral blood mononuclear cells (PBMC) (1 × 10⁵/well) were incubated in quadruplicate with: (i) HIVA peptide pools 1-4; (ii) medium alone; (iii) positive control peptides; and (iv) phytohaemagglutinin 5 μg/ml. CD8 depletion of PBMC was achieved with Dynabeads (M450-CD8; Dynal Biotech Ltd., UK) according to the manufacturer's instructions. Spot-forming units (SFU) were counted with an automated ELISPOT plate reader (AID Systems, Germany). The frequencies of HIV-1-specific IFN-γ-releasing cells were expressed as IFN-γ SFU/million PBMC, after the subtraction of SFU values in negative control wells. Validation criteria were negative control responses of less than 55 SFU/million PBMC and a strongly positive response to phytohaemagglutinin. These were met in more than 96% of assays.

All 10 subjects who enrolled in the study completed the immunization schedule and follow-up. There was minimal or no reaction at the injection site in any subject after the administration of pTHr.HIVA. No systemic reactions nor serious adverse events were observed, and no subject developed anti-DNA antibodies post-immunization. The viral load remained less than 400 copies/ml at all post-immunization timepoints tested in all but one subject, whose viral load was 942 copies/ml on a single visit (day 14), possibly as a result of reduced therapy adherence. CD4 T-cell counts were maintained at or above baseline levels in all vaccinees.

We evaluated responses to HIVA peptide pools with undepleted and CD8-depleted PBMC at the timepoints indicated (Fig. 1). At baseline, we detected a response to at least one HIVA peptide pool in nine of the study participants. The depletion of CD8 T cells abrogated the total gag response by more than 80% in five out of 10 subjects; in the remainder, the gag response was low (< 200 SFU) in undepleted PBMC (data not shown). Overall, there was no significant change in the total gag peptide-specific response from baseline to 4 weeks after the second DNA immunization (day 0 versus day 49, P = 0.26, Wilcoxon signed rank test); responses decreased at days 77 and 105 but returned to baseline by one year. However, in three individuals transiently increased responses to one or more peptide pools, by at least twofold, were observed post-vaccination (data not shown).

The finding that virus-specific CD8 and CD4 T-cell responses were amplified in only a minority of the vaccinees is not entirely unexpected because DNA vaccination was poorly immunogenic in macaques ^[3]. It is possible that a higher response rate or responses of greater magnitude could be achieved in humans with higher doses of DNA, or with a live recombinant viral vector boost or the simultaneous administration of cytokine-expressing plasmids ^[4,5].

The design of this study differs from that of previously reported trials ^[6,7], thereby limiting comparison of the data, in that we evaluated our DNA vaccine in chronically infected patients (median pre-HAART CD4 cell nadir 155 cells/μl) who had received at least one year of HAART. Other studies have included untreated subjects with low viral loads and high CD4 cell counts or subjects who initiated HAART with a high CD4 cell count ^[6]. It is possible that the poor immune responses seen here reflect the relatively advanced stage of disease of our study participants. The late initiation of HAART has been shown to result in impaired responses to tetanus and diphtheria vaccines, even when normal CD4 T-cell counts were attained ^[8]. Early HAART was associated with preserved HIV-specific CD4 T-cell responses in two uncontrolled studies ^[9,10].

In conclusion, this DNA vaccine, pTHr.HIVA, was safe in chronically infected HAART-treated individuals, but its capacity to boost virus-specific CD4 and CD8 T-cell responses was poor. This and other studies ^[6,11,12] have suggested that DNA vaccines are unlikely to have sufficient potency for use as a single modality immunization strategy. However, in a recent study of SIV-infected macaques the immunogenicity of a therapeutic recombinant fowlpox vaccine was enhanced by priming with DNA ^[13]. The effect of previous immunization with pTHr.HIVA on cellular immune responses stimulated by a live recombinant HIVA-expressing vaccine are therefore being evaluated.

Acknowledgements

The authors are indebted to the volunteers for their participation. In addition, they would like to thank Tim Rostron for HLA typing and Carey Lewis for administrative and clinical support.

Sponsorship: This work was funded by the Medical Research Council, UK, and Objectif Recherche Vaccins SIDA (ORVACS). L.D. is the recipient of an MRC Clinician Scientist Fellowship.