Characterization of T-Cell Responses to Conserved Regions of the HIV-1 Proteome in BALB/c Mice

Abstract

A likely requirement for a protective vaccine against human immunodeficiency virus type 1 (HIV-1)/AIDS is, in addition to eliciting antibody responses, induction of effective T cells. To tackle HIV-1 diversity by T-cell vaccines, we designed an immunogen, HIVconsv, derived from the most functionally conserved regions of the HIV-1 proteome and demonstrated its high immunogenicity in humans and rhesus macaques when delivered by regimens combining plasmid DNA, nonreplicating simian (chimpanzee) adenovirus ChAdV-63, and nonreplicating modified vaccinia virus Ankara (MVA) as vectors. Here, we aimed to increase the decision power for iterative improvements of this vaccine strategy in the BALB/c mouse model. First, we found that prolonging the period after the ChAdV63.HIVconsv prime up to 6 weeks increased the frequencies of HIV-1-specific, gamma interferon (IFN-γ)-producing T cells induced by the MVA.HIVconsv boost. Induction of strong responses allowed us to map comprehensively the H-2d-restricted T-cell responses to these regions and identified 8 HIVconsv peptides, of which three did not contain a previously described epitope and were therefore considered novel. Induced effector T cells were oligofunctional and lysed sensitized targets in vitro. Our study therefore provides additional tools for studying and optimizing vaccine regimens in this commonly used small animal model, which will in turn guide vaccine improvements in more expensive nonhuman primate and human clinical trials.

Early Kinetics of the HLA Class I-Associated Peptidome of MVA.HIVconsv-Infected Cells

“…The PBMCs were thawed, and an equal volume of RPMI supplemented with 10% FCS (RPMI10) plus 50 U/ml Benzonase nuclease (Novagen) warmed to 37°C was added dropwise. Cells were expanded by using pools 1 to 6 of HIVconsv-derived 15-mer peptides overlapping by 11 amino acids (aa) ( 22 ) for 10 days, rested, and tested by a gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay as described previously ( 27 ). Thus, ELISPOT plates (catalog number S5EJ044I10; Merck Millipore) prewetted for 1 min with 15 μl of 35% ethanol were coated overnight at +4°C with anti-IFN-γ antibody (10 μg/ml in PBS) (clone 1-D1K; Mabtech).…”

Increased Valency of Conserved-mosaic Vaccines Enhances the Breadth and Depth of Epitope Recognition

“…Cytokine production by splenocytes from immunized mice was assessed by intracellular cytokine staining as described previously. 52 Briefly, splenocytes were stimulated individual peptides for 90 minutes at 37 °C, and then for further 5 hours in the presence of brefeldin A (Golgiplug, BD Biosciences) to prevent cytokine secretion. Cells were surface stained with anti-CD4-Pacific blue, anti-CD8-PE-Texas red (both eBioscience) antibodies and LIVE/DEAD fixable aqua dead cell stain (Invitrogen), and then permeabilized and incubated with various combinations of anti-IL-2-fluoroscein isothiocyanate, anti-IL-4-PE, anti-TNF-α-PE and anti-IFN-γ-APC monoclonal antibodies (Biolegend).…”

“…Killing assay was performed as described previously. 52 Equal numbers of naive HHD splenocytes were differentially labeled with either 800 or 32 nmol/l of CFSE according to the manufacturer's specifications. Splenocytes from immunized mice were prepared as described above, mixed with the differentially CFSE-labeled target cells at an effectorto-target (ET) ratio of 10:1 or 5:1 and incubated overnight at 37 °C.…”

Prospects for a Globally Effective HIV-1 Vaccine

“…Conserved sequences and immune responses have been characterized in animals [147,148], conferring partial protection against SIVmac251 in macaques [149]. In humans, a combination of DNA, ChAd63 and MVA vectors was found safe [150] and immunogenic, inducing high levels of effector T cells that recognized virus-infected autologous CD4+ cells.…”