The importance of adjuvant formulation in the development of a tuberculosis vaccine

Abstract

An effective protein-based vaccine for tuberculosis will require a safe and effective adjuvant. There are few adjuvants in approved human vaccines, including alum and the oil-in-water-based emulsions MF59 (Novartis Vaccines and Diagnostics), AS03 and AS04 (GlaxoSmithKline Biologics), AF03 (Sanofi), and liposomes (Crucell). When used with pure, defined proteins, both alum and emulsion adjuvants are effective at inducing primarily humoral responses. One of the newest adjuvants in approved products is AS04, which combines monophosphoryl lipid A, a TLR-4 agonist, with alum. In this study, we compared two adjuvants: a stable oil-in-water emulsion (SE) and a stable oil-in-water emulsion incorporating glucopyranosyl lipid adjuvant, a synthetic TLR-4 agonist (GLA-SE), each together with a recombinant protein, ID93. Both the emulsion SE and GLA-SE adjuvants induce potent cellular responses in combination with ID93 in mice. ID93/SE induced Th2-biased immune responses, whereas ID93/GLA-SE induced multifunctional CD4(+) Th1 cell responses (IFN-γ, TNF-α, and IL-2). The ID93/GLA-SE vaccine candidate induced significant protection in mice and guinea pigs, whereas no protection was observed with ID93/SE, as assessed by reductions in bacterial burden, survival, and pathology. These results highlight the importance of properly formulating subunit vaccines with effective adjuvants for use against tuberculosis.

Figure 1

Increased ID93-specific IgG2c endpoint titers are induced following mouse immunization with ID93/GLA-SE; increased IgG1 titers are induced with ID93/SE. Serum was collected two weeks after the first immunization (Day 14) or two weeks after the last immunization (Day 56). Mean reciprocal dilutions are represented as the endpoint titer (Log10) ± SD. Asterisks represent statistical significance, where p<0.05, comparing ID93/SE to ID93/GLA-SE. A two-tailed Mann Whitney t-test was used to determine significance. This is one representative study of at least two independent studies.

Figure 2

ID93/GLA-SE increases IFN-γ responses whereas ID93/SE increases IL-5 responses. Mice were immunized 3 times, 3 weeks apart and spleens were harvested from individual mice (n=3/group) one week following the last immunization. The frequency of (A) IFN-γ, or (B) IL-5 secreting T-cells was determined by ELISPOT. Results are represented as spot forming units/million cells ± SD. The asterisks indicate statistical significance compared to saline, where p<0.05. This is one representative study of at least two independent studies.

Figure 3

ID93/GLA-SE induces polyfunctional CD4⁺ T-cells (IFN-γ, TNF-α, IL-2). Cytokine production from ID93-specific CD4⁺ T-cells in immunized mice (n=3 individual mice/group) was measured by flow cytometry. Splenocytes from vaccinated mice were stimulated with ID93 for 12 hs in the presence of GolgiStop. ID93-stimulated splenocytes were identified by ICS based on CD3 and CD4 expression and were further gated on CD44^high cells. The results are represented as the percent frequency of cells expressing triple cytokines (IFN-γ, TNF-α and/or IL-2) double cytokines, or single cytokines ± SD for each of the groups. The asterisks indicate significance by students t-test, where p<0.05 of ID93/SE compared to ID93/GLA-SE. The number sign indicates significance by students t-test compared to saline, where p<0.05. This is one representative study of at least two independent studies.

Figure 4

ID93-specific IgG1, IgG2 and total IgG endpoint antibody titers in guinea pigs following a boost immunization (Day 42). (A) IgG1, (B) IgG2, (C) Total IgG. For statistical analysis, *p<0.05 compared to saline.

Figure 5

ID93-specific proliferative responses in guinea pig PBMCs following immunization with either ID93/SE or ID93/GLA-SE. PBMCs were isolated 3 weeks after the third immunization and frozen. Thawed PBMCs were labeled with Cell Proliferation Dye eFluor670, cultured for 5 days with the indicated stimulators, stained with anti-CD4-RPE and anti-CD8-FITC antibodies and dead cells labeled with propidium iodide (PI). Proliferation of CD4⁺ or CD8⁺ lymphocytes were defined as a decrease in eFluor670 fluorescence intensity in live cells and expressed as either % of live CD4⁺ cells or % of live CD8⁺ cells ± SEM.(A) Gating strategy; (B) Percent proliferation of CD4⁺ or CD8⁺ T-cells from stimulated PBMCs. An additional study was performed in guinea pigs using the same adjuvants with an ID83 subunit vaccine which resulted in similar results.

Figure 6

ID93/GLA-SE confers long-term protection against Mtb in guinea pigs. Guinea pigs were injected with saline or were immunized with BCG, ID93/SE, or ID93/GLA-SE. The data is represented as percentage survival of guinea pigs over time following challenge with Mtb to Day 210, at which point all saline-treated guinea pigs succumbed to infection. Log-rank test was used for statistical comparisons of median guinea pig survival among the experimental groups. P values ≤0.05 were considered significant. An additional study was performed in guinea pigs using the same adjuvants with an ID83 subunit vaccine which resulted in similar results.

Figure 7

Histology of representative lung lobes from each group. Lungs were stained with Masson’s Trichrome stain (1X magnification). Collagen (indicative of lung fibrosis) is stained blue, the cell nuclei are stained black, and the cytoplasm stains red. The dark purple areas indicate lung consolidation. (A) Saline, lung from guinea pig euthanized on Day 135; (B) BCG, Day 234 (collected at the end of study); (C) ID93/SE, guinea pig euthanized on Day 143; (D) ID93/GLA-SE; Day 234 (collected at the end of study).