Enhanced immunogenicity of an HIV-1 DNA vaccine delivered with electroporation via combined intramuscular and intradermal routes

Abstract

It is accepted that an effective prophylactic HIV-1 vaccine is likely to have the greatest impact on viral transmission rates. As previous reports have implicated DNA-priming, protein boost regimens to be efficient activators of humoral responses, we sought to optimize this regimen to further augment vaccine immunogenicity. Here we evaluated single versus concurrent intradermal (i.d.) and intramuscular (i.m.) vaccinations as a DNA-priming strategy for their abilities to elicit humoral and cellular responses against a model HIV-1 vaccine antigen, CN54-gp140. To further augment vaccine-elicited T and B cell responses, we enhanced cellular transfection with electroporation and then boosted the DNA-primed responses with homologous protein delivered subcutaneously (s.c.), intranasally (i.n.), i.m., or transcutaneously (t.c.). In mice, the concurrent priming regimen resulted in significantly elevated gamma interferon T cell responses and high-avidity antigen-specific IgG B cell responses, a hallmark of B cell maturation. Protein boosting of the concurrent DNA strategy further enhanced IgG concentrations but had little impact on T cell reactivity. Interestingly protein boosting by the subcutaneous route increased antibody avidity to a greater extent than protein boosting by either the i.m., i.n., or t.c. route, suggesting that this route may be preferential for driving B cell maturation. Using an alternative and larger animal model, the rabbit, we found the concurrent DNA-priming strategy followed by s.c. protein boosting to again be capable of eliciting high-avidity humoral responses and to also be able to neutralize HIV-1 pseudoviruses from diverse clades (clades A, B, and C). Taken together, we show that concurrent multiple-route DNA vaccinations induce strong cellular immunity, in addition to potent and high-avidity humoral immune responses.

Importance: The route of vaccination has profound effects on prevailing immune responses. Due to the insufficient immunogenicity and protection of current DNA delivery strategies, we evaluated concurrent DNA delivery via simultaneous administration of plasmid DNA by the i.m. and i.d. routes. The rationale behind this study was to provide clear evidence of the utility of concurrent vaccinations for an upcoming human clinical trial. Furthermore, this work will guide future preclinical studies by evaluating the use of model antigens and plasmids for prime-boost strategies. This paper will be of interest not only to virologists and vaccinologists working in the HIV field but also to researchers working in other viral vaccine settings and, critically, to the wider field of vaccine delivery.

FIG 1

Concurrent priming vaccination with EP elicits stronger cellular and higher-avidity antibody responses. (a and b) Single i.d. (10 μg) or i.m. (50 μg) vaccination was compared to concurrent i.d. and i.m. (10 μg and 50 μg) vaccination (n = 8 mice per group), in the absence of EP, for the elicited antigen-specific serum IgG antibody (a) and cellular (b) responses. Humoral and cellular responses were evaluated 1 week after the fourth and final vaccination by antigen-specific IgG ELISA and IFN-γ ELISpot assay and are shown as group means (±SEM). (c to e) Comparison of the ability of different concurrent vaccination regimens, in the presence or absence of EP, to elicit antigen-specific serum (μg/ml) (c) and mucosal (ng/ml) (d) IgG responses and to stimulate systemic cellular immunity (numbers of SFU per million antigen-stimulated cells [±SEM]) (e). Mice (n = 8 per group) were vaccinated 4 times at 3-week intervals, with bleeds occurring 1 week after each vaccination. Mucosal sampling occurred during study week 10, 1 week after the final vaccination. Statistical significance was assessed using the Mann-Whitney U test. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005; N.S., not significant. (f) To assess antibody avidity, concurrent vaccination serum samples were prediluted to give an optical density of between 1 and 1.5 using an in-house antigen-specific ELISA. Samples were then titrated in an endpoint ELISA in duplicate using nonreducing (PBS) and reducing (8 M urea) washes after sample addition. Results are shown as the percent change in binding [(reducing OD/nonreducing OD) × 100].

FIG 2

The route of administration of protein boost impacts the ensuing serum humoral responses. (a and b) Antigen (Ag)-specific IgG (a) and IgA (b) from serum were assessed (n = 8 per group) 1 week after three concurrent DNA-priming vaccinations (i.d. [10 μg] and i.m. [50 μg]) and two protein boosts (20 μg/dose). Protein boosts were delivered by either the s.c., i.n., t.c., or i.m. route. Antibody results are expressed as group means (μg/ml or ng/ml). Statistical significance was assessed using the Mann-Whitney U test. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005. (c) To assess antibody avidity, vaccine serum samples were prediluted to give an optical density between 1 and 1.5 using an in-house antigen-specific ELISA. Samples were then titrated in an endpoint ELISA in duplicate using nonreducing (PBS) and reducing (8 M urea) washes after sample addition. Results are shown as the percent change in binding [(reducing OD/nonreducing OD) × 100].

FIG 3

The route of protein boost vaccination does not significantly affect the magnitude of the cellular response. Vaccinated mice (n = 8 per group) were sacrificed 1 week after three concurrent DNA-priming vaccinations (i.d. [10 μg] and i.m. [50 μg]) and two protein boosts (20 μg/dose). Protein boosts were delivered by either the s.c., i.n., t.c., or i.m. route. Splenocytes were assessed by IFN-γ ELISpot assay for antigen-reactive T cells using two sets of peptide pools consisting of 15-mers overlapping by 11 amino acids (a and b). Graphs are expressed as group means (numbers of SFU per million antigen-stimulated cells [±SEM]). Statistical significance was assessed using the Mann-Whitney U test. ***, P ≤ 0.0005.

FIG 4

Concurrent DNA priming (EP) and recombinant protein boost elicits strong humoral responses with virus-neutralizing activity. (a) Antigen-specific IgG from serum was assessed (n = 6 NWZ rabbits per group) by an in-house ELISA 1 week after either three concurrent DNA-priming vaccinations (i.d. [20 μg] and i.m. [100 μg]) and two protein boosts (40 μg/dose), three concurrent DNA-priming vaccinations (i.d. [20 μg] and i.m. [100 μg]), or two protein boosts. Antibody results are expressed as group means (±SEM) (μg/ml). (b) To assess antibody avidity, rabbit serum samples were prediluted immediately after DNA priming and after two protein boost vaccinations to give an optical density of between 1 and 1.5 using an in-house antigen-specific ELISA. Samples were then titrated in an endpoint ELISA in duplicate using nonreducing (PBS) and reducing (8 M urea) washes after sample addition. Results are shown as the percent change in binding [(reducing OD/nonreducing OD) × 100[. (c) TZM.bl virus neutralization assays were performed on week 13 serum samples using tier 1 pseudoviruses from clades A (DJ263.8), B (SF162.LS), and C (MW965.26) and MuLV as a control. The ID₅₀ titer was calculated as the serum dilution that caused a 50% reduction in the numbers of RLUs compared to those for the virus control wells (TZM.bl cells with virus) after subtraction of the number of RLUs for the cell control (TZM.bl cells alone). Statistical significance was assessed using the Mann-Whitney U test. *, P ≤ 0.05; **, P ≤ 0.005.