Inhibition of elastase enhances the adjuvanticity of alum and promotes anti–SARS-CoV-2 systemic and mucosal immunity

Significance We report that suppression of the serine protease elastase reshapes innate responses induced by injected vaccines containing alum adjuvant. This reprogramming improves the induction of protective antibodies in the bloodstream and stimulates innate signals, which support the development of antibody responses in mucosal tissues. Our findings identify elastase as the innate regulator that blunts the adjuvant activity of alum. They also demonstrate that vaccination via mucosal routes is not an absolute requirement for antibody responses in mucosal tissues and secretions. Supplementation of an alum-based vaccine containing SARS-CoV-2 spike protein subunit 1 as antigen increased anti–SARS-CoV-2 immunity in the blood and mucosal secretions in mice. Thus, this strategy could help in the development of future protein-based vaccines against SARS-CoV-2.


Animals
Specific pathogen-free (SPF) wild-type C57BL/6J mice and Elane tm1Sds/ (ELANE KO) mice which lack elastase were obtained from Jackson Laboratory (Bar Harbor, ME). Caspase 1 KO mice were obtained from (Dr. Dixit, Genentech). All mice were maintained at the Ohio State University animal care facility and were provided food and drink ad libitum. Porcine spleens were specimens from White-Duroc crossbred pigs raised in the BSL2 facility at OARDC and used as controls for other unrelated studies. All animal experiments were approved by the OSU Animal Care and Use Committee.

Patient samples and specimens.
All samples were deidentified specimens from a clinical laboratory, and handling of these samples was under an approved IRB protocol (OSU 2020H0228). Plasma and serum were collected from hospitalized COVID-19 inpatients or ICU patients, OSU HCWs, and blinded convalescent plasma donors and analyzed in a blinded manner.
Mice that received the NEI showed no change in their vitality, food consumption and body weight.
Blood, fecal and vaginal wash samples were collected weekly, and nasal washes were collected at the time of euthanasia to monitor serum and mucosal antibody immunoglobulin isotypes and subclass responses.

Evaluation of antigen-specific antibody responses
To determine OVA-specific and PA-specific antibody titers, ELISA was performed as described previously (1-4). Briefly, microtiter plates were coated with OVA (1 mg/ml) or PA (5 µg/ml). For detection of OVA-specific IgG and IgA Abs, serum or fecal material extracts were serially diluted in PBS 1% BSA, added to the plates and the binding antibodies were detected with HRPconjugated anti-mouse gor a-heavy chain-specific antisera (Southern Biotech Associates Inc., Birmingham, AL). Biotin-conjugated rat anti-mouse IgG1, IgG2a/c, IgG2b or IgG3 monoclonal Abs and HRP-conjugated streptavidin (BD Bioscience, san Jose, CA) were used to measure IgG subclass responses. The reactions were revealed by addition of the water-soluble HRP substrate ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt, Sigma-Aldrich) and the Ab titers were determined as the last dilutions of samples with an absorbance of > 0.1 above that of control samples from naïve mice.
For assessment of IgA responses in the intestinal secretions, freshly emitted fecal pellets were normalized by homogenization in PBS (1 ml per 0.1 g feces). After centrifugation, dilutions of supernatants were used for evaluation of antigen-specific IgA levels as described above.

Analysis of total and antigen-specific serum IgE Ab responses.
Total IgE Ab levels were determined by a BD OptEIA Set Mouse IgE, (BD PharMingen) according to instructions from the manufacturer. To prevent interference of IgG in the assay, serial dilutions of immune plasma were previously depleted of IgG by overnight incubation in Reacti-Bind Protein G Coated Plates (Pierce, Rockford, IL) (4). In order to detect antigen-specific IgE, the microtiter plates were coated with OVA (1 mg/mL) or PA (5 µg/ml). Serial dilutions of IgG-depleted plasma were then added and IgE were detected with a biotinylated anti-mouse IgE Ab (BD Biosciences).
The IgE titers were determined as described above for IgG and IgA.

Quantification of high affinity antibody responses
High affinity antibody responses were measured by ELISA as described above with a minor modification. Briefly, plates were coated with PA and incubated with dilutions of the samples. Urea (4 mM) was then added and the plates incubated for 30 min at room temperature to remove antibodies that bound to the antigen with low affinity (5,6). After washing, the detection antibodies were added and the remaining steps of the ELISA conducted as described above.

Assessment of toxin neutralizing antibodies
Toxin neutralization assay was performed as previously described (2, 3, 6, 7). Briefly, sample dilutions were added to J774 macrophages cultured in cultured in RPMI supplemented with 10 % fetal calf serum. Bacillus anthracis lethal toxin (LeTx) [i.e., PA plus Bacillus anthracis lethal factor (LF, List Biological, Campbell, CA)] was then added to the plates. After overnight incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma-Aldrich) was added to assess the viability of macrophages as a function of redox potential. The toxin neutralizing antibody titers were determined as the lowest concentration of serum that protects macrophages from the cytotoxicity of LeTx.

In vivo trafficking of B cells
To identify the mucosal sites of B cell trafficking, we performed adoptive cell transfer. Briefly, Recipient mice were euthanized 18hr later and CFSE + cells present in the spleens or mucosal tissues were analyzed by flow cytometry.

B cell epitope mapping
Array of 181-peptides of 17-or 13-mers, with 10 amino acid overlaps that span the spike (S) glycoprotein of the USA-WA1/2020 (GenPept: QHO60594) of the SARS-CoV-2 (NR-52402, BEI Bioresources) was used to identify linear B cell epitopes recognized by anti-SARS Spike protein S1 antibodies. Briefly, microtiter plates were coated with individual peptide (20 µg/ml). Samples were then added and the binding antibodies were detected with HRP-conjugated anti-mouse g-specific antisera or biotin-conjugated rat anti-mouse IgG1 or IgG2a/c followed by HRP-conjugated streptavidin. For identification of linear B cell epitopes recognized antibody in the sera of COVID-19 patients, the binding antibodies were detected with HRP-conjugated anti-human IgG. To show the location of epitopes on protein, epitopes were labeled on 3D structure of SARS-CoV-2 spike protein (PDB ID: 6ZOW) by using iCn3D web-based 3D structure viewer provided from NCBI.

Analysis of antigen-specific T helper cell cytokine responses and expression of homing receptors
Antigen-specific T helper cell cytokine responses were analyzed by flow cytometry after in vitro restimulation and intracellular staining with cytokine-specific fluorescent antibodies. Briefly, splenocytes and mesenteric lymph nodes were collected on day 28 after the first immunization and restimulated with antigen (i.e., 1 mg/ml of OVA or 15 µg/ml of PA) in vitro as previously

Real-time RT-PCR.
Tissues were collected, snap frozen, and reduced to powder before adding TRIzol (Invitrogen, Carlsbad, CA). Complementary DNA was synthesized using Superscript III (Invitrogen). Realtime RT-PCR was performed as previously described (1) using the following primers:

Quantification of porcine IgM, IgG, and IgA.
Porcine IgM, IgG, and IgA were measured using an ELISA using immunoglobulin standards and anti-pig IgM, IgG, and IgG antibodies (Bio-Rad).

SARS-CoV-2 pseudovirus neutralization assay.
For determination of virus neutralizing activity, lentiviral SARS-CoV-2 pseudotyped virus was constructed and used as described in the previous study (8). Specifically, 100 µL of virus were incubated with sera or nasal wash solutions for 1 h at 37C and the mixture was added to