Severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology

Abstract

Although the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathology. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addition of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were associated with significantly increased lung eosinophilic immunopathology on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathology by vaccines containing delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathology predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the critical importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses.

Importance: Coronaviruses such as SARS-CoV and Middle East respiratory syndrome-associated coronavirus (MERS-CoV) cause high case fatality rates and remain major human public health threats, creating a need for effective vaccines. While coronavirus antigens that induce protective neutralizing antibodies have been identified, coronavirus vaccines present a unique problem in that immunized individuals when infected by virus can develop lung eosinophilic pathology, a problem that is further exacerbated by the formulation of SARS-CoV vaccines with alum adjuvants. This study shows that formulation of SARS-CoV spike protein or inactivated whole-virus vaccines with novel delta inulin-based polysaccharide adjuvants enhances neutralizing-antibody titers and protection against clinical disease but at the same time also protects against development of lung eosinophilic immunopathology. It also shows that immunity achieved with delta inulin adjuvants is long-lived, thereby overcoming the natural tendency for rapidly waning coronavirus immunity. Thus, delta inulin adjuvants may offer a unique ability to develop safer and more effective coronavirus vaccines.

FIG 1

Adjuvant effects on humoral immunity. (A) Female BALB/c mice were immunized i.m. twice with 1 μg rSP alone or with Advax-1, Advax-2, or CpG and then periodically bled for measurement of serum SP-specific antibodies by ELISA. (B) At 2 weeks and 1 year postimmunization, mice (n = 6 for each time point) were sacrificed and bone marrow collected for measurement of memory B-cell frequency by ELISPOT assay. (C) Also shown are changes of serum SP-specific IgG titers over time. All values are means ± standard errors of the means (SEM). #, group where there was a significant (P < 0.05) fall in antibody titer between 2 weeks and 1 year postimmunization. *, P < 0.05; **, P < 0.01; ***, P < 0.005), ****, P < 0.001.

FIG 2

Adjuvant effects on T-cell immunity. (A and B) At 2 weeks and 1 year postimmunization, splenocytes were harvested from immunized mice (n = 6 for each time point, divided into 2 groups of 3 mice) for rSP-stimulated T-cell proliferation by CFSE assay (A) and cytokine ELISPOT assays (B). (C) Representative FACS CFSE plot of the data in panel A, demonstrating reduced CD4 and CD8 T-cell proliferation in response to SP in the CpG group. (D) Representative IFN-γ ELISPOT assays showing that the majority of SP-specific IFN-γ producing splenocytes in immunized mice recognize the CD4 epitope peptide from SP (left well) rather than the CD8 epitope peptide. All values are means ± SEM.

FIG 3

Adjuvant effects on T-cell cytokine responses. Splenocytes were harvested at 2 weeks and 1 year postimmunization and stimulated with rSP for 2 days, and cytokines in the culture supernatants were measured by cytometric bead array (CBA) assay. Values are means ± SEM.

FIG 4

Vaccine protection and serum antibody titers. Groups of mice (n = 15) were immunized twice i.m. with vehicle, Advax-1 or -2 adjuvant alone, rSP alone or formulated with alum, Advax-1, or Advax-2, or IWV alone or with Advax-2 and then at 4 weeks postimmunization challenged with SARS-CoV. Shown are survival curves (A), serum SARS-CoV-specific IgG titers (B), and serum SARS-CoV neutralization titers (C). All values are means ± SEM.

FIG 5

Vaccine effects on SARS-CoV lung pathology and virus titers. Groups of immunized mice (n = 5 for each time point) were sacrificed on days 3 and 6 postchallenge and lungs harvested. Shown are lung histology scores (A and B), lung weights (C and D), lung virus titers (limit of detection, 0.75) (E and F), and SP-specific lung IgG titers (G and H). All values are means ± SEM.

FIG 6

Lung eosinophil infiltration on day 6 postchallenge. The lungs of immunized mice from each group with the highest eosinophil infiltration scores on H&E staining on day 6 postchallenge were stained with a specific eosinophil MBP antibody (brown color).