Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

Abstract

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Fig. 1. : co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) enhances human immunodeficiency virus-1 subtype B conserved peptides (HIVBr18)-induced T-cell responses. Two weeks after the last immunisation with HIVBr18 co-administrated with pVAX1 or pGM-CSF, pooled spleen cells from six BALB/c mice were cultured in the presence of both pooled and individual HIVBr18-encoded peptides (5 µM), or medium only. Frequencies of interferon (IFN)-γ-secreting T-cells against pooled (A) and individual (B) HIVBr18-encoded peptides were measured by ELISPOT and shown as spot forming units (SFU) per 106 cells. Mean plus standard deviation of three independent experiments are shown in A. One representative experiment of three is shown in B. Only peptides frequently recognised are shown. Dotted line represents ELISPOT cut-off (15 SFU/106 cells). Asterisks mean p < 0.001.

Fig. 2. : co-administration of pasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) increases the frequency of human immunodeficiency virus-1 subtype B conserved peptides (HIVBr18)-induced polyfunctional CD4+ T-cells. Two weeks after the last immunisation with HIVBr18 co-administrated with pVAX1 or pGM-CSF, pooled spleen cells from six BALB/c mice were collected, labelled with carboxyfluorescein succinimidyl ester (CFSE) (1.25 mM) and cultured for four days in the presence of pooled HIVBr18-encoded peptides or medium only. On day 4, cells were pulsed for 12 h with pooled peptides in the presence of Brefeldin A and co-stimulatory antibody (anti-CD28), and then stained for CD3, CD4, CD8, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, and interleukin (IL)-2. Multiparameter flow cytometry strategy used to determine the frequency of polyfunctional T-cells (A). Frequency of cytokine producing CFSElow CD4+ T-cells (B) and CD8+ T-cells (D). Frequency of CFSElow CD4+ (C) and CD8+ (E) T-cells producing all the combinations of IFN-γ, TNF-α, and IL-2. Background responses were subtracted in each condition. One representative experiment of three is shown in B-E. APC: antigen presenting cell; FITC: fluorescein isothiocyanate; FSC: forward scatter; SSC: side scatter; *: p < 0.05; **: p < 0.01; ***: p < 0.001.