Multivalent TB vaccines targeting the esx gene family generate potent and broad cell-mediated immune responses superior to BCG

Abstract

Development of a broad-spectrum synthetic vaccine against TB would represent an important advance to the limited vaccine armamentarium against TB. It is believed that the esx family of TB antigens may represent important vaccine candidates. However, only 4 esx antigens have been studied as potential vaccine antigens. The challenge remains to develop a vaccine that simultaneously targets all 23 members of the esx family to induce enhanced broad-spectrum cell-mediated immunity. We sought to investigate if broader cellular immune responses could be induced using a multivalent DNA vaccine representing the esx family protein members delivered via electroporation. In this study, 15 designed esx antigens were created to cross target all members of the esx family. They were distributed into groups of 3 self-processing antigens each, resulting in 5 trivalent highly optimized DNA plasmids. Vaccination with all 5 constructs elicited robust antigen-specific IFN-γ responses to all encoded esx antigens and induced multifunctional CD4 Th1 and CD8 T cell responses. Importantly, we show that when all constructs are combined into a cocktail, the RSQ-15 vaccine, elicited substantial broad Ag-specific T cell responses to all esx antigens as compared with vaccination with BCG. Moreover, these vaccine-induced responses were highly cross-reactive with BCG encoded esx family members and were highly immune effective in a BCG DNA prime-boost format. Furthermore, we demonstrate the vaccine potential and immunopotent profile of several novel esx antigens never previously studied. These data highlight the likely importance of these novel immunogens for study as preventative or therapeutic synthetic TB vaccines in combination or as stand alone antigens.

Keywords: BCG; DNA vaccines; TB vaccine; electroporation; esx family; prime-boost; tuberculosis.

Figure 1. Construction and expression of esx constructs. (A) Schematic representation of all 5 trivalent esx constructs encompassing a total of 15 esx antigens. All genes are cloned into the pVAX1 mammalian vector and are under the CMV promoter. The N-terminal IgE leader peptide, C-terminal HA tag, BGH polyA signal, kanamycin resistance gene and pUC origin are shown. (B) Expression of esx constructs in RD cells as analyzed by western blot analysis detected using an anti-HA mAb. Also shown is a loading control by staining for actin and relative sizes are indicated (KDa).

Figure 2. Immunogenicity of esx constructs compared. (A) Immunization schedule for DNA. Mice (n = 5 per group) were vaccinated plus EP with each individual DNA esx construct 3 times at 2-wk intervals and spleens were harvested 1 wk after last immunization to analyze the cellular immune responses (B–F). Esx-specific T cell responses in (B–F) were measured against a pool of peptides to their respective antigens by IFN-γ ELISpot. Error bars indicate SEM and experiments were performed independently at least 2 times with similar results.

Figure 3. Evaluation of the esx-specific CD4 and CD8 T cells responses following DNA vaccination. CB6F1 mice (n = 5) were immunized by i.m./EP with 3 injections at 2-wk intervals with 20 µg of each individual esx construct. Splenocytes were collected 1 wk after final vaccination, stimulated with their respective peptide pools and analyzed by flow cytometry following intracellular staining using antibodies against IFN-γ and TNF-α. (A) The gating strategy used to analyze the frequency of CD4 and CD8 T cells positive for IFN-γ and TNF-α cytokines. (B) Column graphs depicting esx-specific CD4 T cells releasing the cytokines IFN-γ (C) TNF-α and (D) double-positive producing cells (and pVAX control). (E) Column graphs show the esx-specific CD8 T cells releasing the cytokines IFN-γ (F) TNF-α and (G) double-positive producing cells (and pVAX control). Background staining from cells stimulated with medium alone has been subtracted. Error bars represent SEM of 5 mice per group. Experiments were performed independently at least 2 times with similar results.

Figure 4. Immunization with RSQ-15 induces broader and stronger esx-specific Th1 immune responses compared with BCG. (A) Immunization schedule for DNA (black line) and BCG vaccination (blue line). CB6F1 mice (n = 5) were immunized 3 times at 2 wk intervals with all esx constructs co-delivered as a cocktail (RSQ-15 vaccine; 20 ug per esx construct) and 1 mo later, T cell responses were analyzed using splenocytes. (B) The frequency of esx-specific IFN-γ producing cells determined by IFN-γ ELISpot assay. (C) BCG mice were immunized by a single s.c. BCG injection (10⁶ CFU) and splenocytes from BCG-primed mice were stimulated with all individual esx-specific peptide pools and IFN-γ production measured by ELISpot assay. Error bars indicate SEM and experiments were performed independently at least 2 times with similar results.

Figure 5. Prime-boost BCG vaccination with RSQ-15 DNA vaccine increases the esx-specific BCG-induced responses. (A) Immunization schedule for the 2 different prime-boost regimens. CB6F1 mice were immunized s.c. with 10⁶ CFU of BCG SSI. Six weeks later, mice were boosted i.m once (1x) or boost immunized twice (2x) at 2 wk intervals with 100 µg (20 µg per esx construct) of the RSQ-15 vaccine. Seven days after the 1× (B) and 2× (C) boost, spleens were assayed by IFN-γ ELISpot. pVAX1 and BCG-only controls were included. Results represent SEM of 5 mice per group. Experiments were performed independently at least 2 times with similar results.

Figure 6. Esx DNA constructs elicit esx-specific cross-reactivity among their subfamily ortholog members. Mice were immunized with 20 µg of pORF or pVSW 3 times at 2 wk intervals and spleens were harvested 1 wk later and then stimulated with their respective esx-specific peptide pools to monitor the degree of cross-reactivity between esx antigens determined by IFN-γ ELISpot (A) Recognition of esxV, esxH, esxN, and esxL after pORF vaccination. (B) Recognition of esxO, esxK, esxP, esxM, and esxG after pVSW vaccination. Error bars indicate SEM and data shown are representative of 5 mice per group in 2 independent experiments that generated similar results.