key: cord-0878652-gksk1g21 authors: Zhu, Jingen; Jain, Swati; Sha, Jian; Batra, Himanshu; Ananthaswamy, Neeti; Kilgore, Paul B.; Hendrix, Emily K.; Hosakote, Yashoda M.; Wu, Xiaorong; Olano, Juan P.; Kayode, Adeyemi; Galindo, Cristi L.; Banga, Simran; Drelich, Aleksandra; Tat, Vivian; Tseng, Chien-Te K.; Chopra, Ashok K.; Rao, Venigalla B. title: A bacteriophage-based, highly efficacious, needle and adjuvant-free, mucosal COVID-19 vaccine date: 2022-04-29 journal: bioRxiv DOI: 10.1101/2022.04.28.489809 sha: 607ea9070a97ae9f9b4edf9be1ad76ab9ef3d62e doc_id: 878652 cord_uid: gksk1g21 The authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected and effective in preventing COVID-19, but do not induce efficient mucosal immunity, or prevent viral transmission. We developed a bacteriophage T4-based, multicomponent, needle and adjuvant-free, mucosal vaccine by engineering spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of T4-COVID vaccine induced higher virus neutralization antibody titers against multiple variants, balanced Th1/Th2 antibody and cytokine responses, stronger CD4+ and CD8+ T cell immunity, and higher secretory IgA titers in sera and bronchoalveolar lavage with no effect on the gut microbiota, compared to vaccination of mice intramuscularly. The vaccine is stable at ambient temperature, induces apparent sterilizing immunity, and provides complete protection against original SARS-CoV-2 strain and its Delta variant with minimal lung histopathology. This mucosal vaccine is an excellent candidate for boosting immunity of immunized and/or as a second-generation vaccine for the unimmunized population. immunity, or prevent viral transmission. We developed a bacteriophage T4-based, 23 multicomponent, needle and adjuvant-free, mucosal vaccine by engineering spike 24 trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal 25 administration of T4-COVID vaccine induced higher virus neutralization antibody titers 26 against multiple variants, balanced Th1/Th2 antibody and cytokine responses, stronger 27 CD4 + and CD8 + T cell immunity, and higher secretory IgA titers in sera and 28 bronchoalveolar lavage with no effect on the gut microbiota, compared to vaccination of 29 mice intramuscularly. The vaccine is stable at ambient temperature, induces apparent 30 sterilizing immunity, and provides complete protection against original SARS-CoV-2 strain 31 and its Delta variant with minimal lung histopathology. This mucosal vaccine is an 32 excellent candidate for boosting immunity of immunized and/or as a second-generation 33 vaccine for the unimmunized population. (Tiboni et al., 2021) . 58 The current vaccines developed using the spike protein of the ancestral SARS-CoV-2 59 strain (Wuhan-Hu-1) show progressively diminished efficacy against the subsequently 60 emerged viral variants of concern (VOCs) such as Alpha, Beta, Gamma, Delta, and most 61 4 recently Omicron and its subvariant BA.2, which are more efficiently transmitted and/or 62 more lethal. The evolutionary space for emergence of newer SARS-CoV-2 63 variants/subvariants that are even more efficiently transmissible and also more lethal 64 that might render the current vaccines ineffective remains a worrisome and real 65 possibility (Markov et al., 2022) . 66 Considering the evolutionary path of the virus, the most desired next-generation 67 vaccine(s) would be one that can induce strong mucosal immunity, in addition to broader pandemic. Additionally, platforms that are needle-and adjuvant-free and stable at 75 ambient temperatures would greatly accelerate global distribution efforts, not only for 76 controlling the current COVID-19 pandemic but also for any future epidemic or pandemic. 77 Furthermore, needle-free vaccines can be administered easily and safely, and may 78 provide the best option to vaccinate children. 79 We recently reported (Zhu et al., 2021 ) the development of a "universal" phage T4 rapidly generate multivalent vaccine candidates. Using an intramuscular immunization 83 scheme, an optimal COVID-19 vaccine candidate (referred to as T4-CoV-2) was selected 84 that elicited robust immunogenicity, virus neutralizing activity, and complete protection 85 against ancestral SARS-CoV-2 challenge in a mouse model. This vaccine consisted of T4 86 phage decorated with ~100 copies of prefusion-stabilized spike ectodomain trimers (S-87 trimers) on the surface of 120 x 86 nm virus capsid (Fig. 1A ). In addition, the vaccine also 88 contained SARS-CoV-2 nucleocapsid protein (NP) packaged in the capsid core and a 12-89 amino acid (aa) peptide of the putative external domain of E protein (Ee) fused to the 90 highly antigenic outer capsid protein (Hoc) displayed on the capsid surface (Fig. 1A) . 91 The protective immunity of the T4-CoV-2 nanovaccine could potentially be because (Yao et al., 2020) . Therefore, we hypothesized that it is probable that such a T4-97 CoV-2 nanoparticle when exposed to nasal mucosal surfaces might be recognized as a 98 natural viral intruder by the resident immune cells, stimulating strong mucosal as well as 99 systemic immune responses ( Figures 1B to 1D) . Furthermore, the S-trimer-displayed T4- Figure S1 ). The phosphate-buffered saline (PBS) 163 and T4-vetor control groups, as expected, induced no significant antigen-specific 164 antibodies, whereas the T4-CoV-2 vaccinated groups (either i.m. or i.n.) triggered high 165 levels of IgG antibodies ( Figures 2C and 2F) . 166 High levels of both Th1 (IgG2a) and Th2 (IgG1) subtype antibodies were induced by 167 i.m. and i.n. immunizations, demonstrating that the T4- CoV-2 vaccine triggered balanced 168 9 Th1-and Th2-derived antibody responses (Figures 2D, 2E, 2G, and 2H ). This is in contrast 169 to the alum-adjuvanted subunit vaccines that show strong Th2-bias (Zhu et al., 2021) . The 170 balanced immune response was also uniformly recapitulated in a dose response 171 experiment. Nearly the same levels of Th1 and Th2 antibody responses were elicited with 172 the medium-dose as with the high-dose, while the levels were lower (5-25-fold) with the 173 low-dose or single-dose antigen ( Figure S1 ). 174 Intriguingly, the T4-CoV-2 vaccine induced high levels of spike-specific serum IgA IFNγ is a predominant cytokine secreted by effector CD8 + T cells, Th1 CD4 + T cells, 210 and NK cells (Castro et al., 2018) . More specifically, with re-stimulation of splenocytes 211 using S protein, significant levels of IFNγ + CD8 + cells, which play a critical role in SARS- 212 CoV-2 viral clearance, were observed in i.n.-immunized mice ( Figure 2L ). Additionally, 213 significantly elevated percentages of CD4 + T cells producing IFNγ were detected in the i.n. 214 group in comparison to the i.m. group of vaccinated mice ( Figure 2M , Figure S2C ). These 215 data indicated an enhanced Th1-mediated immunity induced by i.n. administration of the 216 vaccine. Of note, we did not observe significant differences between i.n. and i.m. routes 217 of immunization regarding either the IFNγ + CD8 + cells or the IFNγ + CD4 + cells when 218 restimulated with S-and NP-peptides ( Figures S2D and S2E ). Probably the conformational 219 epitopes in S-and NP-proteins could contribute to these differences of higher IFNγ levels Figure S3B ). In addition, the T4-CoV-2-β 404 vaccine also contained ~100 copies of NP protein packaged inside the capsid ( Figure S3C ). 405 Five-week-old hACE2 AC70 mice were immunized with this vaccine using the same prime- The Tukey mean comparison method between the T4-597 CoV-2 and PBS groups for the top four genera (Alistipes, Muribaculaceae, Clostridiales, 598 and Anaeroplasma) indicated no significant differences in the gut microbiota even 599 though there were few differences in numbers (e.g., for Alistipes and Muribaculaceae) 600 when vaccine was administered by the i.n. route ( Figure 7G ). However, a significant 601 difference in the Muribaculaceae genus was noted when T4-CoV-2 vaccine was delivered 602 by the i.m. route ( Figure 7H ). These same differences were observed among the 603 Bacteroidetes phylum indicating that i.m. administration of the T4-CoV-2 vaccine had a 604 more significant impact on the gut microbiota. These trends were also reflective 605 upstream of the hierarchy from families to the phylum of the recovered gut microbiota. antigens per capsid were calculated using gp23 (major capsid protein; 930 copies) or gp18 778 (major tail sheath protein; 138 copies) as internal controls and S-trimer protein standard. 779 The copies of the phage-packaged NP protein were quantified by Western blotting using 780 the commercial rabbit anti-NP antibody (Sino Biological) and NP protein standard 781 (ThermoFisher Scientific) as previously described (Zhu et al., 2021) . 100% controls. Two sets of the same phages were stored at 4°C or 22°C, and samples 785 were taken at two-week intervals for ten weeks and were flash-frozen at -70°C. All the 786 samples were thawed and analyzed together for stability and functionality by SDS-PAGE 787 and human ACE2 receptor binding assay as previously described (7). After Coomassie 788 Blue R-250 (Bio-Rad) staining and destaining, the displayed S-trimer protein bands on 789 SDS-PAGE gels were scanned and quantified by ChemiDoc MP imaging system (Bio-Rad) 790 and ImageJ. 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All animal experiments were approved by the 833 days 0 (prime) and 21 (boost), while for 1-dose regimen, the vaccine was given at day 21. 834 Three different number of phage particles possessing 0.8, 4.8, and 20 µg of S-trimer 835 antigens representing ~ 1.0 x 10 10 , 6 x 10 10 and 2.5 x 10 11 phage particles, respectively, 836 were used. Negative control mice received the same volume of PBS or the same amount 837 of T4 control phage (T4 control). Blood was drawn from each animal on day 0 (pre-bleed) 838 and day 42, the isolated sera were stored at −80°C until further use.