A synthetic adjuvant to enhance and expand immune responses to influenza vaccines

Rhea N Coler, Susan L Baldwin, Narek Shaverdian, Sylvie Bertholet, Steven J Reed, Vanitha S Raman, Xiuhua Lu, Joshua DeVos, Kathy Hancock, Jacqueline M Katz, Thomas S Vedvick, Malcolm S Duthie, Christopher H Clegg, Neal Van Hoeven, Steven G Reed, Rhea N Coler, Susan L Baldwin, Narek Shaverdian, Sylvie Bertholet, Steven J Reed, Vanitha S Raman, Xiuhua Lu, Joshua DeVos, Kathy Hancock, Jacqueline M Katz, Thomas S Vedvick, Malcolm S Duthie, Christopher H Clegg, Neal Van Hoeven, Steven G Reed

Abstract

Safe, effective adjuvants that enhance vaccine potency, including induction of neutralizing Abs against a broad range of variant strains, is an important strategy for the development of seasonal influenza vaccines which can provide optimal protection, even during seasons when available vaccines are not well matched to circulating viruses. We investigated the safety and ability of Glucopyranosyl Lipid Adjuvant-Stable Emulsion (GLA-SE), a synthetic Toll-like receptor (TLR)4 agonist formulation, to adjuvant Fluzone® in mice and non-human primates. The GLA-SE adjuvanted Fluzone vaccine caused no adverse reactions, increased the induction of T helper type 1 (T(H)1)-biased cytokines such as IFNγ, TNF and IL-2, and broadened serological responses against drifted A/H1N1 and A/H3N2 influenza variants. These results suggest that synthetic TLR4 adjuvants can enhance the magnitude and quality of protective immunity induced by influenza vaccines.

Conflict of interest statement

Competing Interests: Dr. Steven G. Reed is a founder of, and holds an equity interest in, Immune Design Corporation, a licensee of certain rights associated with GLA. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. GLA-SE enhances HI titers to…
Figure 1. GLA-SE enhances HI titers to the A/H3N2 vaccine component and antigenically drifted A/H3N2 influenza strains.
Balb/c mice were immunized with Fluzone (2006–2007) either alone ( - ), or formulated with emulsion (SE), or glucopyranosyl-lipid-adjuvant (GLA-SE). (A) Serum HI titers one week after the prime and boost (N = 7 mice/group). Shown are the individual mouse HI titers (diamonds) and the geometric mean titer (GMT) (indicated by the horizontal bars±the 95% CI) against the vaccine strain (Wis/05) and the antigenically drifted H3N2 strains. The assay detection limit was set at ≤10. Statistical significance between groups was determined and compared to Fluzone (*p

Figure 2. GLA-SE enhances IgG-secreting BMPCs to…

Figure 2. GLA-SE enhances IgG-secreting BMPCs to vaccine and drifted A/H3N2 strains and induces cross-reactive…

Figure 2. GLA-SE enhances IgG-secreting BMPCs to vaccine and drifted A/H3N2 strains and induces cross-reactive type-1 immunity.
Mice were immunized Fluzone (2006–2007) either alone ( - ), or formulated with emulsion (SE), or GLA-SE. (A) Shown are virus-specific [vaccine (Wis/05) or drifted H3N2 strains] IgG-secreting BMPC responses five weeks after boost from individual mice (diamonds; N = 4/group). The group mean is represented by the horizontal bar ± standard deviation (s.d.) per million cells. Statistical significance was determined and compared to Fluzone (*p

Figure 3. GLA-SE enhances serum HI titers…

Figure 3. GLA-SE enhances serum HI titers to the A/H1N1 and A/H3N2 components in Fluzone.

Figure 3. GLA-SE enhances serum HI titers to the A/H1N1 and A/H3N2 components in Fluzone.
NHPs (N = 3/group) were injected with saline (-), or were immunized with Fluzone (2007–2008) alone (+), or Fluzone formulated with emulsion (SE), or GLA-SE (1, 5, 25 or 50 µg). Serum samples were collected 4 weeks after the prime and boost immunizations, at day 30 or day 58, respectively. Shown are the HI titers from individual animals (diamonds) and GMT (indicated by the horizontal bars ± the 95% CI) against the three components of the Fluzone vaccine.

Figure 4. GLA-SE enhances serum HI titers…

Figure 4. GLA-SE enhances serum HI titers to antigenically drifted A/H1N1 and A/H3N2 influenza strains…

Figure 4. GLA-SE enhances serum HI titers to antigenically drifted A/H1N1 and A/H3N2 influenza strains following a boost immunization.
NHPs (N = 3/group) were immunized with Fluzone (2007–2008) alone, or formulated with emulsion (SE), or GLA-SE (1, 5, 25 or 50 µg). Serum samples were collected 4 weeks after the boost (day 58). Shown are the HI titers from individual animals (diamonds) and the GMT (indicated by the horizontal bars ± the 95% CI) against the antigenically A/Brisbane/59/2007(H1N1), A/New Caledonia/20/99(H1N1) and A/Beijing/262/1995(H1N1) strains and the drifted A/Uruguay/716/2007(H3N2) strain.

Figure 5. Cellular responses after injection with…

Figure 5. Cellular responses after injection with saline, Fluzone alone, or combined with SE or…

Figure 5. Cellular responses after injection with saline, Fluzone alone, or combined with SE or GLA-SE.
NHP (N = 3/group) were given 2 injections, 4 weeks apart with saline, Fluzone, Fluzone+SE or Fluzone+GLA-SE (1, 5, 25 and 50 µg). (A) IFNγ, IL-2, TNF and IL-5 (pg/mL) recall responses after stimulation of whole blood with H1N1 or H3N2 were evaluated by CBA at day 0 and day 58. (B) IFNγ:IL-5 ratios of cytokine recall responses after stimulation of whole blood with H1N1 or H3N2, were evaluated by CBA at day 58. (C) Granzyme B levels (pg/mL) responses in whole blood following stimulation with H1N1 or H3N2 was evaluated by Luminex at day 58. Horizontal lines represent median Granzyme B concentrations. (D) Chemokine responses to Fluzone antigens after immunization. MCP-3 and IP-10 (pg/mL) responses in whole blood following stimulation with H1N1 or H3N2 were evaluated by Luminex at day 58. Horizontal lines represent median chemokine concentrations.
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References
    1. Katz JM, Veguilla V, Belser JA, Maines TR, Van Hoeven N, et al. The public health impact of avian influenza viruses. Poult Sci. 2009;88:872–879. - PubMed
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Figure 2. GLA-SE enhances IgG-secreting BMPCs to…
Figure 2. GLA-SE enhances IgG-secreting BMPCs to vaccine and drifted A/H3N2 strains and induces cross-reactive type-1 immunity.
Mice were immunized Fluzone (2006–2007) either alone ( - ), or formulated with emulsion (SE), or GLA-SE. (A) Shown are virus-specific [vaccine (Wis/05) or drifted H3N2 strains] IgG-secreting BMPC responses five weeks after boost from individual mice (diamonds; N = 4/group). The group mean is represented by the horizontal bar ± standard deviation (s.d.) per million cells. Statistical significance was determined and compared to Fluzone (*p

Figure 3. GLA-SE enhances serum HI titers…

Figure 3. GLA-SE enhances serum HI titers to the A/H1N1 and A/H3N2 components in Fluzone.

Figure 3. GLA-SE enhances serum HI titers to the A/H1N1 and A/H3N2 components in Fluzone.
NHPs (N = 3/group) were injected with saline (-), or were immunized with Fluzone (2007–2008) alone (+), or Fluzone formulated with emulsion (SE), or GLA-SE (1, 5, 25 or 50 µg). Serum samples were collected 4 weeks after the prime and boost immunizations, at day 30 or day 58, respectively. Shown are the HI titers from individual animals (diamonds) and GMT (indicated by the horizontal bars ± the 95% CI) against the three components of the Fluzone vaccine.

Figure 4. GLA-SE enhances serum HI titers…

Figure 4. GLA-SE enhances serum HI titers to antigenically drifted A/H1N1 and A/H3N2 influenza strains…

Figure 4. GLA-SE enhances serum HI titers to antigenically drifted A/H1N1 and A/H3N2 influenza strains following a boost immunization.
NHPs (N = 3/group) were immunized with Fluzone (2007–2008) alone, or formulated with emulsion (SE), or GLA-SE (1, 5, 25 or 50 µg). Serum samples were collected 4 weeks after the boost (day 58). Shown are the HI titers from individual animals (diamonds) and the GMT (indicated by the horizontal bars ± the 95% CI) against the antigenically A/Brisbane/59/2007(H1N1), A/New Caledonia/20/99(H1N1) and A/Beijing/262/1995(H1N1) strains and the drifted A/Uruguay/716/2007(H3N2) strain.

Figure 5. Cellular responses after injection with…

Figure 5. Cellular responses after injection with saline, Fluzone alone, or combined with SE or…

Figure 5. Cellular responses after injection with saline, Fluzone alone, or combined with SE or GLA-SE.
NHP (N = 3/group) were given 2 injections, 4 weeks apart with saline, Fluzone, Fluzone+SE or Fluzone+GLA-SE (1, 5, 25 and 50 µg). (A) IFNγ, IL-2, TNF and IL-5 (pg/mL) recall responses after stimulation of whole blood with H1N1 or H3N2 were evaluated by CBA at day 0 and day 58. (B) IFNγ:IL-5 ratios of cytokine recall responses after stimulation of whole blood with H1N1 or H3N2, were evaluated by CBA at day 58. (C) Granzyme B levels (pg/mL) responses in whole blood following stimulation with H1N1 or H3N2 was evaluated by Luminex at day 58. Horizontal lines represent median Granzyme B concentrations. (D) Chemokine responses to Fluzone antigens after immunization. MCP-3 and IP-10 (pg/mL) responses in whole blood following stimulation with H1N1 or H3N2 were evaluated by Luminex at day 58. Horizontal lines represent median chemokine concentrations.
Figure 3. GLA-SE enhances serum HI titers…
Figure 3. GLA-SE enhances serum HI titers to the A/H1N1 and A/H3N2 components in Fluzone.
NHPs (N = 3/group) were injected with saline (-), or were immunized with Fluzone (2007–2008) alone (+), or Fluzone formulated with emulsion (SE), or GLA-SE (1, 5, 25 or 50 µg). Serum samples were collected 4 weeks after the prime and boost immunizations, at day 30 or day 58, respectively. Shown are the HI titers from individual animals (diamonds) and GMT (indicated by the horizontal bars ± the 95% CI) against the three components of the Fluzone vaccine.
Figure 4. GLA-SE enhances serum HI titers…
Figure 4. GLA-SE enhances serum HI titers to antigenically drifted A/H1N1 and A/H3N2 influenza strains following a boost immunization.
NHPs (N = 3/group) were immunized with Fluzone (2007–2008) alone, or formulated with emulsion (SE), or GLA-SE (1, 5, 25 or 50 µg). Serum samples were collected 4 weeks after the boost (day 58). Shown are the HI titers from individual animals (diamonds) and the GMT (indicated by the horizontal bars ± the 95% CI) against the antigenically A/Brisbane/59/2007(H1N1), A/New Caledonia/20/99(H1N1) and A/Beijing/262/1995(H1N1) strains and the drifted A/Uruguay/716/2007(H3N2) strain.
Figure 5. Cellular responses after injection with…
Figure 5. Cellular responses after injection with saline, Fluzone alone, or combined with SE or GLA-SE.
NHP (N = 3/group) were given 2 injections, 4 weeks apart with saline, Fluzone, Fluzone+SE or Fluzone+GLA-SE (1, 5, 25 and 50 µg). (A) IFNγ, IL-2, TNF and IL-5 (pg/mL) recall responses after stimulation of whole blood with H1N1 or H3N2 were evaluated by CBA at day 0 and day 58. (B) IFNγ:IL-5 ratios of cytokine recall responses after stimulation of whole blood with H1N1 or H3N2, were evaluated by CBA at day 58. (C) Granzyme B levels (pg/mL) responses in whole blood following stimulation with H1N1 or H3N2 was evaluated by Luminex at day 58. Horizontal lines represent median Granzyme B concentrations. (D) Chemokine responses to Fluzone antigens after immunization. MCP-3 and IP-10 (pg/mL) responses in whole blood following stimulation with H1N1 or H3N2 were evaluated by Luminex at day 58. Horizontal lines represent median chemokine concentrations.

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