A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice

Abstract

DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans.

Figure 1. Vaccine constructs and Tat expression.

(A) Schematic representation of the vaccine constructs; (B) reducing Western blot analysis of Tat in cell lysates from HEK293T cells transfected with plasmid DNA encoding the different forms of Tat (tracks 2–5) and (C) non-reducing Western blot analysis of Tat in supernatant fluids of HEK293T cells transfected with pVAX-Tat-IMX313 (track 2) or pVAX-sTat-IMX313 (track 3) DNA. The 42 kDa β-actin protein was used as loading control for the Western blot. Blots in Fig. 1B,C are cropped for clarity and conciseness; full length blots of these figures are presented in supplementary figure S1A,B.

Figure 2. Tat DNA vaccination induces Th cell responses and humoral immunity.

Mice (n = 7 per group) were vaccinated ID with 3 doses of 50 μg of pVAX, pVAX-Tat, pVAX-Tat-IMX313, pVAX-sTat or pVAX-sTat-IMX313 at 2 week intervals. Splenocytes were collected 14 days after the last dose. Blood was collected one day before each vaccination and on the day of euthanasia. (A) IFN-γ ELIspot assay to examine Tat-specific CMI in splenocytes stimulated with Tat peptides for 36 h. (B) FTA assay to examine Th cell responses from pVAX-Tat, pVAX-Tat-IMX313, pVAX-sTat or pVAX-sTat-IMX313 vaccinated mice above the GMFI of CD69 on FTA B220⁺ cells from naïve mice. The graph is representative of the FTA with cells pulsed with Tat peptide pools 1, 2 or 3. (C) AUC values for Th cell responses depicted in (B). (D) The results of an indirect ELISA to detect Tat-specific serum IgG. Titres are expressed as the reciprocal of the serum dilution and plotted as log10 IgG endpoint titre. The data are representative of 2 independent experiments in which each serum sample was analysed in duplicate. (E) Tat transactivation activity in Cf2-Luc cells after the addition of Tat pre-incubated in PBS or a 1/25 dilution of serum samples from mice vaccinated with the respective vaccines. Graphs show mean RLUs (±SEM) relative to untransfected control cells and are representative of 3 independent experiments in which each sample was analysed in triplicate. (F) Percentage decline in luminescence (an indicator of the neutralisation of Tat transactivation activity) depicted in (E). Data shown in the entire figure depict the mean (n = 7) ± SEM; an unpaired non-parametric Mann–Whitney U test was used to analyse the statistical significance of the data; *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001 and p ≥ 0.005 = non-significant (ns).

Figure 3. Humoral responses and CMI are increased after 5 doses of Tat DNA vaccine.

Animals were vaccinated ID with 5 doses of 50 μg of pVAX-sTat or pVAX-sTat-IMX313 at 2 week intervals. Splenocytes were collected 14 days after the last dose. Blood and CVLs were collected one day before each vaccination and on the day of euthanasia. (A) ELISA results showing serum anti-Tat IgG titres. (B) Serum neutralisation of Tat transactivation activity, (C) Percentage decline in luminescence (an indicator of the neutralisation of Tat transactivation activity) that is depicted in (B,D) ELISA results showing anti-Tat sIgA titres in CVLs from pVAX-sTat or pVAX-sTat-IMX313 vaccinated mice. Data are representative of 2 independent experiments. (E) Percentage of vaccinated animals in which sIgA was detected after 3–5 vaccine doses in the experiment shown in 3D. (F) IFN-γ ELIspot assay to examine Tat-specific CMI responses following administration of 5 doses of pVAX-sTat or pVAX-sTat-IMX313. Data shown in the entire figure depict the mean (n = 7) ± SEM; an unpaired non-parametric Mann–Whitney U test was used to analyse the statistical significance of the data; *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001 and p ≥ 0.005 = non-significant (ns).

Figure 4. pVAX-sTat-IMX313 vaccinated mice exhibit superior control against EcoHIV challenge.

Unvaccinated mice or vaccinated mice which received 5 doses of 50 μg of either pVAX-sTat or pVAX-sTat-IMX313 were challenged with 1.5 μg p24 of EcoHIV/NL4-3 10 days post final vaccination. The viral load in PECs and splenocytes was determined by qRT-PCR; EcoHIV RNA levels in (A) PECs and (B) splenocytes 7 days post challenge. EcoHIV mRNA levels were normalised to RPL13a mRNA and the data represent the mean (n = 7) ± SEM. **p < 0.05 and **p ≤ 0.01(Mann–Whitney U test).