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

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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.

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The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) was identified in 2003 when a series of fatal pneumonia cases started in Hong Kong (1, 2). SARS-CoV lung infection is characterized by a marked inflammatory cell infiltrate with diffuse alveolar damage (3). Before the disease was controlled by quarantine measures, ∼8,000 humans were clinically infected, with an overall case fatality rate of ∼10% but with mortality of ∼50% in those over 65 years of age (4). After recovery from coronavirus infections, previously infected individuals may be susceptible to reinfection (5, 6). In fact, individuals with waning immunity may be at risk of even more severe disease upon coronavirus reexposure (7). Given the risk of future human outbreaks of SARS-CoV, Middle East respiratory syndrome-associated coronavirus (MERS-CoV) (8), or other coronaviruses, development of an optimal vaccine platform is an ongoing priority.

SARS-CoV is a positive-stranded RNA virus 29.7 kb in length with approximately 14 open reading frames (9). The first SARS-CoV vaccine candidates were produced from inactivated SARS-CoV. Inactivated whole virus (IWV) without adjuvant provided only modest protection, inducing low neutralizing-antibody titers and earlier lung clearance in challenged ferrets (10). In mice, IWV vaccines alone or formulated with alum adjuvant provided partial protection, but this was associated with severe eosinophilic lung pathology (11,–14), similar to the pathology seen with SARS-CoV rechallenges after primary infection (15). It is not known if immunized human subjects might similarly be predisposed to lung immunopathology upon SARS-CoV infection. Another challenge for inactivated SARS-CoV vaccines is the need for biosafety level 3 (BSL3) facilities for vaccine manufacture. An alternative vaccine antigen is the SARS-CoV spike protein (SP), which mediates target cell entry via attachment to angiotensin-converting enzyme 2 and CD209L, leading to receptor-mediated endocytosis (16, 17). While immunization with recombinant SP (rSP) induced protection (18, 19), it similarly exacerbated lung eosinophilic immunopathology, and like with IWV vaccine, this was exacerbated by formulation with alum adjuvant (11, 14). Respiratory syncytial virus (RSV) vaccines formulated with alum similarly caused lung eosinophilic immunopathology and increased mortality when immunized children became infected with RSV (20), suggesting that this is a generalized problem of Th2-polarizing alum adjuvants. There is a critical need, therefore, to identify suitable coronavirus vaccine adjuvants that do not exacerbate, or ideally suppress, virus-induced lung immunopathology. Advax belongs to the class of polysaccharide adjuvants (21, 22) and is based on particles of β-d-[2-1]poly(fructo-furanosyl)α-d-glucose (delta inulin) (23). Delta inulin has been shown to enhance humoral and cellular immunity with a wide variety of vaccines against viruses, including influenza virus (24, 25), Japanese encephalitis and West Nile viruses (26, 27), hepatitis B virus (28), and HIV (29). It induces balanced Th1 and Th2 immune responses (25, 28), which contrasts with alum's marked Th2 bias. Delta inulin is notable for its lack of reactogenicity as demonstrated in human influenza and hepatitis B vaccine trials (30, 31).

This study asked whether given its balanced effects on Th1 and Th2 T-cell immunity, the Advax delta inulin adjuvant platform could be used to enhance SARS-CoV vaccine protection while avoiding the risk of lung eosinophilic immunopathology. As shown below, delta inulin adjuvants successfully enhanced humoral and cellular immunity and protection against SARS-CoV while avoiding the lung immunopathology induced by alum adjuvant.

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Recombinant spike protein (rSP) produced in insect cells (BEI catalog no. NR-722; Protein Sciences Corp., Meriden, CT) and Vero cell formaldehyde- and UV light-inactivated whole-virion SARS-CoV (BEI catalog no. NR-3882, lot 480405CA) were both obtained from BEI Resources, NIAID, NIH (Manassas, VA). CpG2006 was purchased from GeneWorks (Adelaide, Australia). Advax adjuvants were supplied by Vaxine Pty Ltd., Adelaide, Australia, with Advax-1 being a preservative-free sterile suspension of delta inulin microparticles at 50 mg/ml in a bicarbonate buffer, whereas Advax-2 additionally included 10 μg CpG per 1 mg delta inulin. Both formulations of Advax adjuvant were used at a standardized dose of 1 mg delta inulin per mouse. Advax adjuvant was formulated with antigen by simple admixture immediately prior to immunization.



Vaccine immunogenicity studies were performed in accordance with the Animal Experimentation Guidelines of the National Health and Medical Research Council of Australia, approved by the Flinders Animal Welfare Committee, and performed on adult female BALB/c mice 6 to 8 weeks of age as supplied by the Flinders University animal facility. Mice were immunized twice 3 weeks apart with a variety of different vaccine formulations and then bled regularly for 12 months to monitor changes in antibody levels.

The SARS challenge study was conducted in accordance with and with the approval of the Institutional Animal Care and Use Committee of Utah State University. The work was done in the AAALAC-accredited Laboratory Animal Research Center of Utah State University in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (2010 revision). Female 4- to 6-week-old BALB/c mice weighing 18 to 20 g were obtained from Charles River Laboratories (Wilmington, MA). They were maintained on Wayne Lab Blox and tap water ad libitum. At days 3 and 6 after virus challenge, 5 mice from each immunized and control group were sacrificed and the lungs harvested for gross pathology (lung score), lung weights, lung virus titers, and measurement of anti-SARS IgG in lung homogenate.



Mouse-adapted SARS-CoV was used throughout the study. The virus used for the challenge study was passaged 25 times through BALB/c mice and was verified as SARS-CoV by enzyme-linked immunosorbent assay ELISA and PCR (2). The virus was sent for sequencing and identified as a SARS-CoV variant as described elsewhere (32).



SARS-specific antibodies were determined by ELISA. rSP was absorbed to ELISA plates in 0.1 M sodium hydrogen carbonate buffer, pH 9.6, and incubated overnight at 4°C. After blocking with 1% bovine serum albumin-phosphate-buffered saline (BSA-PBS) for 1 h, serum samples diluted in 1% BSA-PBS were incubated for 2 h at room temperature (RT) and washed, and then 100 μl biotinylated anti-mouse IgG, IgG1, IgG2a, IgG2b, IgG3, or IgM antibodies (Abcam) with streptavidin-horseradish peroxidase (HRP) (BD Biosciences) was incubated for 1 h at RT and then incubated with 100 μl of tetramethylbenzidine (TMB) substrate for 10 min; the reaction was then stopped with 1 M phosphoric acid. The optical density at 450 nm (OD450) was measured with a VersaMax ELISA microplate reader (Molecular Devices, CA, USA) and analyzed using SoftMax Pro software.


T-cell assays.

Mice were killed by cervical dislocation, and bones and spleens were collected. Bone marrow was isolated from femurs by flushing with 3% fetal bovine serum (FBS)-PBS. Cells were released by pressing against a tea strainer with a rubber syringe plunger, and red blood cells (RBCs) were removed by osmotic shock. Cells were washed with 3% FBS-PBS and then resuspended in RPMI complete medium with 10% heat-inactivated FBS. Splenocytes were labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen Life Technologies) and cultured in 24-well plates at 106 cells/ml/well with 1 μg/ml rSP. After 5 days of incubation at 37°C and 5% CO2, cells were washed with 0.1% BSA-PBS, treated with anti-mouse CD16/CD32 (BD Biosciences) for 5 min at 4°C, and then stained with anti-mouse CD4-allophycocyanin (APC) and anti-mouse CD8a-phycoerythrin (PE)-Cy7 (BD) for 30 min at 4°C. Cells were washed and then analyzed by fluorescence-activated cell sorting (FACS) (FACSCanto II; BD Biosciences) with FACSDiva software. For each lymphocyte subset, proliferation was expressed as the percentage of divided cells (CFSE low) compared to undivided cells (CFSE high). Dot plots representing analysis of 105 cells were generated by FlowJo software.


ELISPOT assays.

The frequency of antigen-specific antibody- or cytokine-secreting cells was analyzed using biotinylated anti-mouse IgG, IgG1, IgG2a, or IgM antibodies (Abcam), anti-mouse gamma interferon (IFN-γ), interleukin-2 (IL-2), or IL-4 antibody pairs (BD), or LEAF anti-mouse IL-17A and biotin-anti-mouse IL-17A antibody (BioLegend, USA) with streptavidin-HRP (BD Biosciences), according to the manufacturer's instructions. Briefly, single-cell suspensions were prepared from bone marrow and spleens of mice at the indicated time points and plated at 2 × 105 cells/well in 96-well plates precoated with rSP (for antibody detection) or anti-mouse cytokine monoclonal antibody (MAb) (for cytokine detection) overnight at 4°C and then blocked with RPMI–10% FBS. For cytokine assays, the cells were incubated with rSP (10 μg/ml) at 37°C and 5% CO2 for 2 days. Wells were washed, incubated with biotinylated anti-mouse Ig or anti-mouse cytokine MAb at RT for 2 h, and washed again, and then streptavidin-HRP was added and left for 1 h before washing and addition of aminoethylcarbazole (AEC) substrate solutions (BD Biosciences). Spots were counted with an ImmunoSpot S6 enzyme-linked immunosorbent spot (ELISPOT) analyzer (CTL, USA) and analyzed using ImmunoSpot software. The number of spots in negative-control wells was subtracted from the number of spots in SP wells, and the results were expressed as antibody-secreting cells (ASC) per 106 bone marrow cells or spots per 106 splenocytes.


Lung studies.

Immunized and vehicle-treated mice were sacrificed at days 3 and 6 after virus challenge, and lungs were removed. Formalin-fixed lungs were mounted in paraffin blocks. Paraffin-embedded lung sections were stained with hematoxylin and eosin (H&E) and a rat monoclonal antibody (Clone MT-14.7) to eosinophil major basic protein (MBP) (Lee Laboratory, Mayo Clinic, AZ) following a standard immunohistochemistry (IHC) procedure. Diaminobenzidine (DAB) chromogen was used to identify eosinophils as brown-stained cells, as previously described (14). Eosinophil infiltration was scored without knowing animal identity using H&E-stained slides. An overall infiltration score (0 to 3) was assigned to each section according to the amounts of eosinophils in the parenchyma and their distributions through the lung, as follows: 0, no to a few eosinophils; 1, mild eosinophil infiltration; 2, moderate infiltration; and 3, severe infiltration. For confirmation, immunohistochemistry to the eosinophil major basic protein was determined in sections with the highest score of each treatment group.


Statistical analysis.

Group comparisons for antibody and ELISPOT tests were done by the Mann-Whitney test. Virus titers were compared using analysis of variance to determine experimental significance, followed by Newman-Keuls pairwise-comparison tests. Survival analysis was done using the Kaplan-Meier method and a log rank test. Pairwise comparisons of survivor curves were analyzed by the Mantel-Cox log rank test, and the relative significance was adjusted to a Bonferroni-corrected significance threshold for the number of treatment comparisons done. Significance is indicated in the figures as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.005; and ****, P < 0.001.

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Vaccine effects on humoral immunity.

To assess the ability of Advax adjuvant formulations to enhance the immunogenicity of recombinant spike protein (rSP), adult female BALB/c mice (n = 6/group) were given two intramuscular (i.m.) immunizations 3 weeks apart of 1 μg rSP alone or formulated with Advax-1 or -2 or CpG adjuvant. Formulation with Advax-1 or -2 significantly enhanced SP-specific immunoglobulin responses compared to immunization with antigen alone. Advax-1 significantly enhanced the IgG1 response at 2 weeks postboost, which was maintained out to 1 year postimmunization (Fig. 1A). In contrast, Advax-2 significantly increased a broad range of antibody isotypes, namely, IgG1, IgG2a, IgG2b, and IgG3, with this pattern being maintained for 1 year postimmunization. CpG adjuvant significantly increased SP-specific IgG2a, IgG2b, and IgG3 but not IgG1 at 2 weeks postimmunization, with this pattern maintained for 1 year postimmunization. SP-specific IgM responses peaked at 2 weeks postimmunization in all groups, but interestingly, they remained significantly higher in both Advax-adjuvanted groups than in the group receiving unadjuvanted rSP alone out to 1 year postimmunization, suggesting the generation of long-lived memory IgM-positive B cells in the Advax-immunized groups. The serological response patterns induced by the different adjuvants were largely mirrored by the frequency of isotype-specific antibody-secreting cells (ASC) in the bone marrow of immunized mice. Advax-1 was exclusively associated with IgG1-positive ASC, whereas Advax-2 was associated with both IgG1- and IgG2a-positive ASC (Fig. 1B). There were 2- to 3-fold more IgM-positive ASC in the Advax-immunized mice at 1 year postimmunization.


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