key: cord-0318185-324sf1zq authors: Paschall, Amy V.; Ozdilek, Ahmet; Briner, Sydney L.; Brindley, Melinda A.; Avci, Fikri Y. title: Modulation of immunosuppressant drug treatment to improve SARS-CoV-2 vaccine efficacy in mice date: 2021-09-30 journal: bioRxiv DOI: 10.1101/2021.09.28.462156 sha: deed709ec25406de5e319d0e993363c52463301e doc_id: 318185 cord_uid: 324sf1zq The COVID-19 pandemic dramatically demonstrated the need for improved vaccination strategies and therapeutic responses to combat infectious diseases. However, the efficacy of vaccines has not yet been demonstrated in combination with commonly used immunosuppressive drug regimens. We sought to determine how common pharmaceutical drugs used in autoimmune disorders can alter immune responses to the SARS-CoV-2 spike protein vaccination. We treated mice with five immunosuppressant drugs (cyclophosphamide, leflunomide, methotrexate, methylprednisolone, and mycophenolate mofetil), each with various mechanisms of action prior to and following immunization with SARS-CoV-2 spike protein. We assessed the functionality of antibody responses to spike protein and compared immune cell populations in mice that received no treatment with those that received continuous or temporarily suspended immune suppressive therapy. All tested immunosuppressants significantly reduced the antibody titers in serum and functional antibody response against SARS-CoV-2 spike protein in immunized mice. Temporarily halting selected immunosuppressants (methylprednisolone and methotrexate, but not cyclophosphamide) improved antibody responses significantly. Through proof-of-principle experiments utilizing a mouse model, we demonstrated that immune suppression in autoimmune disorders through pharmaceutical treatments may impair vaccine response to SARS-CoV-2, and temporary suspension of immunosuppressant treatment may be necessary to mount an effective antibody vaccine response. This work provides feasibility for future clinical assessment of the impact of immunosuppressants on vaccine efficacy in humans. Significance Statement Immunosuppressant regimens are widely used as therapies for a variety of diseases, including autoimmune, inflammatory, and cancer. However, immunosuppressants can impair critical immune responses to vaccination. The impact of standard immunosuppressant use on the critical, developing SARS-CoV-2 vaccination strategies has not been well-described. In this study, we use a mouse model to determine how different immunosuppressant drugs that act through different mechanisms can impair the antibody response to SARS-CoV-2 spike protein, and how modulating these drug regimens may restore antibody levels and function. virus and prevent its infectivity (2) , and are thus a key determinant in determining patient immune protection against SARS-CoV-2. Most current WHO approved vaccines against SARS-CoV-2 mainly target the spike protein. Studies find the vaccines induce robust antibody response as well as T cell responses that are highly effective at preventing severe disease (13) (14) (15) . In addition, treatment with cocktails of monoclonal antibodies against spike are effective in treating COVID-19 patients, suggesting antibody levels are an important component of the immune response to SARS-CoV-2. Patients with autoimmune diseases or other diseases that alter the patient immune landscape have been shown to exhibit poorer responses to SARS-CoV-2 vaccination (16) (17) (18) . Potentially, effective antibody response to SARS-CoV-2 vaccination may be inhibited using immunosuppressants. Immunosuppressants are commonly used in the treatment of autoimmune diseases, including rheumatoid arthritis and lupus, and are also common requirements following organ transplantation. Immunosuppressants target specific or multiple immune cell populations or functional pathways. Furthermore, cancer chemotherapy drugs may induce immune suppression as a secondary effect Approximately 6 million Americans are estimated to be taking an immune-weakening drug (19) . Thus, a large percentage of the population could be expected to generate a weaker immune response following vaccination against COVID- 19. Previous research has shown immunosuppressant treatments can significantly inhibit patient responses to vaccinations against multiple pathogens including viral (20, 21) In this study, we investigated the effects of five widely used immunosuppressant drugs that target different pathways of the immune responses, and if these effects could be modulated following changes to immunosuppressant regimens in mouse models (Table 1) . Specifically, we investigated: cyclophosphamide (CYC, a potent drug known to deplete immune cells, which has been shown to impair vaccine responses (25) ); leflunomide (LEF, a disease-modifying antirheumatic drug, or DMARD, that interferes with immune cell replication and has been shown to inhibit IgE antibody response (26, 27) ); methotrexate (MTX, a potent drug used in multiple disease models which inhibits the activities of multiple enzymes critical for immune cell function and has been shown to interfere in multiple vaccination models (22-24, 28, 29)); methylprednisolone (MP, a commonly used corticosteroid that may alter opsonophagocytic killing of pathogens by immune cells(23)); and mycophenolate mofetil (MM, an inhibitor of purine biosynthesis shown to inhibit antibody responses (30) ). We determined that all drugs significantly reduced antibody response to SARS-CoV-2 spike protein vaccination in mice when administered in doses and routes of administration based on established mouse models (22-30) (Table 1) . However, temporary suspension of drugs at vaccination timepoints may improve vaccine response to the spike protein. These findings may guide clinical studies to demonstrate that immunosuppressant administration can be modulated to improve immune responses generated through vaccinations against COVID-19 in humans. We first sought to determine whether commonly used immune suppressants may dampen the antibody responses to SARS-CoV-2 spike protein, possibly reducing COVID-19 vaccine efficacy. We therefore started mice on immune suppression regimens of CYC, LEF, MM, MP, and MTX, as well as no drug control groups that received phosphate buffered saline (PBS). Each of these immune suppressants has been used in treatment of autoimmune disorders as well as in research of immune responses (Table 1) . To emulate clinical spike protein-based vaccination strategies inducing adaptive humoral immunity, we immunized mouse groups with 0.5 µg/mouse of SARS-CoV-2 recombinantly expressed spike protein in alum, or an alum only control, 7 days after starting treatment regimen (Day 0), and then immunized again 14 days after primary immunization ( Fig 1A) . We first analyzed serum IgG titers at 14 days post-primary and days 17, 21, 28, and 35, which followed booster immunization ( Fig 1B) . All drug treatment groups showed significant decreases in absorbances compared to those of the No Drug control mouse samples. We analyzed individual serum IgG ( Fig 1C) and IgM ( Fig 1D) titers following booster immunization (Day 21) as well as the endpoint of the immunization experiment (Day 35) and observed significant differences in IgG titers in all immunosuppressant-treated mouse groups. Similar trends were observed in IgM titers. These results indicate that immunosuppressant regimens can impair the antibody response to SARS-CoV-2 spike protein vaccination. To determine if this inhibition of IgG and IgM titers against spike protein would translate into functional deficiencies in anti-spike response specifically, we utilized a recombinant vesicular stomatitis virus (rVSV∆G) lacking the VSV glycoprotein and encoding the SARS-CoV-2 spike to monitor virus neutralization (Fig 2) . When comparing 50% neutralization activity 28 days following primary vaccination, we observed the drug treated sera was not as potent as the No Drug control group (No Drug) except for LEF which had no significant differences in activity compared to control. CYC treatment group did not contain any detectable neutralizing activity at any timepoint. For the endpoint (Day 35), on average, MTX required 8 times more sera than the control group to neutralize 50% of the virus and MP treatment required 9 times as much serum (Fig 2B) . We observed functional inhibition of antibody-mediated clearance of spike protein-expressing cells in nearly all of the immunosuppressant-treated groups as compared to control, indicating that continuous immunosuppressant regimens can effectively reduce vaccination response against SARS-CoV-2. We next sought to determine if this inhibition of an effective response to spike protein immunization immunization. We obtained serum at day 14, immediately before the booster immunization, and days 17, 21, 28, and 35, which followed the booster immunization, and performed ELISAs to compare IgG titers in mouse groups that received continuous immunosuppressant treatments through immunization and mouse groups that were temporarily untreated at or around the time of immunization. We observed that all mouse groups that received CYC treatments retained negligible levels of IgG and IgM titers against spike protein, despite interruption of the treatment regimen for both one day and three days of drug suspension ( Fig 3B) . MP, however, exhibited a partial restoration of antibody response as indicated in ELISA, with the highest level of antibody titers observed in mice that did not receive MP two days before, the day of, and two days after each vaccination timepoint ( Fig 3C) . We also observed significant increases in booster titer levels when MTX treatment was halted for 1 or 3 timepoints, compared to continuous MTX treatment. This data indicates that antibody response to SARS-CoV-2 spike protein can be improved through modulation of the immunosuppressant regimen, depending on the drug in use. Furthermore, this translated to functional differences as demonstrated by the level of sera required to neutralize rVSV∆G-SARS-CoV-2-S infectivity (Fig 4) . While halting CYC treatments at or around the time of vaccination did not increase neutralization of the viral particles above the level observed in the continuous CYC treatments (not detectable, Fig 4A) , temporary suspension of MP showed higher neutralization compared to the respective continuous treatments ( Fig 4C) . This indicates that a functional antibody response against the spike protein can be improved through modulation of certain immunosuppressant drugs. While a trend in increased neutralization in the MTX Halt groups (greater than 5X dilution in average comparing Halt X3 to continuous MTX treatment) was observed, this was not considered significant in statistical analysis ( Fig 4B) . In an additional statistical evaluation, effect size calculation was performed with the Halt X1 and X3 groups compared to continuous drug treatments (Supplementary Table 6 ). Halting both MTX and MP for either X1 or X3 timepoints showed very large effect size of the standardized mean differences using Cohen's d calculation compared to the respective continuous drug regimens. These data indicate that a functional antibody response against the spike protein can be improved through modulation of certain immunosuppressant drugs. The immunosuppressants used to treat mice have been associated with altering counts and The COVID-19 pandemic caused by SARS-COV-2 represented a new paradigm of vaccine and immunotherapy design. Vaccine candidates were designed, tested, and approved for human use in an unprecedented timespan. As of July 2021, the WHO has noted 108 vaccine candidates that have progressed to clinical evaluation. While vaccination against SARS-CoV-2 has resulted in dramatic decreases in COVID-19 case numbers, one growing concern has been the effects of immune suppression on these vaccination efforts. We demonstrate here that commonly used immunosuppressants can significantly impair the antibody response to the spike protein of SARS-modulation of the drug treatment. However, many additional considerations should be taken into account for future studies and optimization of these treatment regimens, specifically in humans. One important consideration is the ability of SARS-CoV-2 to evade host immune-mediated cell death mechanisms and promoting inflammatory processes. Research has shown the spike protein The These dosages were selected based on previous established murine research models for each of the respective drug treatments (22-30) to ensure efficacy. Treatments were started at seven days before immunization and administered intraperitoneally (IP). Mice were immunized with SARS-CoV-2 spike protein at 0.5 µg/50µl alum (InvivoGen, stock concentration aluminum 10 µg/µl)/mouse intraperitoneally on days 0 and 14 (Fig 1A) . Alum only mouse groups received 50µl alum alone intraperitoneally. For experiments in which immunosuppressant regimens were temporarily halted at the time of vaccinations, mouse groups received continuous treatments starting at day -8 except at the day of immunization (Halt X1) or except for the two days prior to, two days following, and day of immunization (Halt X3) (Fig 3A) . Individual mouse sera samples (n=4) were collected from mouse groups treated or not treated with immunosuppressants at 17, 21, 28, and 35 days, which followed immunization with spike protein (top panels). 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