key: cord-0736745-c0hfzccj
authors: Counoupas, Claudio; Johansen, Matt D.; Stella, Alberto O.; Nguyen, Duc H.; Ferguson, Angela L.; Aggarwal, Anupriya; Bhattacharyya, Nayan D.; Grey, Alice; Patel, Karishma; Siddiquee, Rezwan; Stewart, Erica L.; Feng, Carl G.; Hansbro, Nicole G.; Palendira, Umaimainthan; Steain, Megan C.; Saunders, Bernadette M.; Low, Jason K. K.; Mackay, Joel P.; Kelleher, Anthony D.; Britton, Warwick J.; Turville, Stuart G; Hansbro, Philip M.; Triccas, James A.
title: A single dose, BCG-adjuvanted COVID-19 vaccine provides sterilizing immunity against SARS-CoV-2 infection in mice
date: 2021-06-03
journal: bioRxiv
DOI: 10.1101/2020.12.10.419044
sha: 096fcdc4e52f986255b896fe33ecf04fba9e51fd
doc_id: 736745
cord_uid: c0hfzccj

Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilized, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralizing antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralized B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.

heterologous protection against disease. For these reasons, a Phase 3, randomised controlled 72 trial in healthcare workers has commenced to determine if BCG vaccination can reduce the 73 incidence and severity of COVID-19 (the BRACE Trial) 9 . While that trial will determine if 74 BCG can reduce the impact on COVID-19 during the current pandemic, BCG does not express 75 SARS-CoV-2 specific antigens and thus, would not induce long-term immune memory. 76

Here, we have exploited the immunostimulatory properties of BCG to develop a SARS-77

CoV-2 vaccine, BCG:CoVac, that combines a stabilized, trimeric form of the spike protein 78 with the alum adjuvant. BCG:CoVac stimulated SARS-CoV-2-specific antibody and T cell 79 responses in mice after a single vaccination, including the elicitation of high-titre NAbs. 80

Critically, a single dose was shown to protect mice against severe SARS-CoV-2, demonstrating 81 that BCG:CoVac is a highly immunogenic and promising vaccine candidate. 82 85 responses in mice 86 The immunostimulatory properties of BCG 11 led to us to test if the vaccine could serve as the 87 backbone for a unique vaccine platform against COVID-19. This was also supported by our 88 observation that prior BCG immunization could augment anti-spike IgG responses after 89 boosting with SpK formulated in Alhydrogel/alum (Alm SpK ) (Fig. S1 ). To determine if this 90 property of BCG could be used in a single vaccine formulation, we subcutaneously (s.c) 91 vaccinated mice with a single dose of BCG formulated with a stabilized, trimeric form of the 92 SARS-CoV-2 spike protein 12 and the titre of IgG2c or IgG1 anti-SpK antibodies was 93 determined at various timepoints post-immunization (Fig. 1a) . While BCG vaccination resulted 94 in background levels of anti-SpK antibodies, titres were approximately 100-fold higher for both 95 antibody isotypes after BCG Spk vaccination, and similar to those levels achieved with Alm SpK 96 ( Fig. 1b, 1c) . Addition of alum to BCG Spk (termed BCG:CoVac) further increased antibodies 97 titres, particularly IgG2c, which were significantly greater after BCG:CoVac vaccination 98 compared to mice immunized with either BCG or Alm SpK , at all timepoints examined (Fig. 1b,  99 1c). 100

The IgG2c Ab isotype correlates with Th1-like immunity in C57BL/6 mice 13 , and such 101 responses are considered necessary for effective protection against SARS-CoV-2 infection 14 . 102 We therefore examined the frequency of IFN-g-expressing T cells after a single dose of 103 BCG:CoVac at 2 weeks post-vaccination. BCG SpK and BCG:CoVac induced the generation of 104 SpK-specific CD4 + and CD8 + T cells secreting IFN-g (Fig. 1d, 1e) , consistent with Th1 105 immunity observed after BCG vaccination 15 . The greatest response was observed after 106 vaccination with BCG:CoVac, with the numbers of IFN-g-secreting T cells significantly 107 increased compared to vaccination with either BCG or Alum SpK . Low levels of the 108 inflammatory cytokines IL-17 and TNF were observed after BCG:CoVac vaccination (Fig. 1e) . 109 We further dissected vaccine-induced immunity by defining the cellular composition in 110 draining lymph nodes 7 days after vaccination. Both Alum SpK CoV-2 vaccines correlate strongly with the induction of neutralizing antibodies (NAbs) 16 . Such 125

NAbs are a key determinant of protection induced by current vaccines used in humans 17 . We 126 therefore measured NAb levels after a single dose of BCG:CoVac. No NAbs were detected in 127 the plasma of mice vaccinated with BCG (Fig. 3a) . Surprisingly, NAb titres were at near 128 background levels for mice vaccinated with BCG SpK (Fig. 3a) , despite the high levels of IgG 129

Ab isotypes detected in these same animals ( Fig. 1) . High NAb titres were detected as early as 130 2 weeks post-immunization upon vaccination with BCG:CoVac, and titres were significantly 131 increased compared to vaccination with Alum SpK (approximate 10-fold increase). The mean 132

NAb titres in the plasma of BCG:CoVac-vaccinated mice were approximately 10-fold greater 133 than those seen in SARS-CoV-2 infected humans (Fig. 3a) . Although the levels of NAbs 134 peaked at 2 weeks post-vaccination with BCG:CoVac, they remained significantly elevated up 135 to day 42 post-immunization unlike those in the other immunized groups. 136

Since previous work suggests that the level of IgG antibody correlates with NAb titres after 137 SARS-CoV-2 infection 18 , we examined whether a similar phenomenon was observed after 138 vaccination with BCG:CoVac. Strong correlation (r> 0.9) was observed between IgG2c isotype 139 and NAbs in groups vaccinated with BCG:CoVac or Alum SpK (Fig. 3b) , with a significant yet 140 less robust correlation between IgG1 and NAbs for these groups (Fig. 3c) . There was no 141 correlation between NAbs and either IgG1 or IgG2c Ab for mice vaccinated with BCG SpK alone 142 ( Fig. 3d, 3e) . 143

These data suggest that alum is required for the optimal generation of NAbs after 144 BCG:CoVac vaccination. This is a significant advantage for implementation of this vaccine 145 candidate, due to the low cost and long standing safety record of alum 19, 20 . Importantly, the 7 potential risk of vaccine-associated enhanced respiratory disease (VAERD) caused by the  147   selective induction of Th2 T cell responses by alum is offset by the strong Th1 immunity  148 induced by BCG:CoVac, which in turn is driven by BCG (Fig. 1e) . showed some beneficial effects and partially protected against weight loss (~10%) and lung 168 IL-6 and KC responses but not in other disease features. Remarkably, vaccination with 169 4b, 4c). These mice had no detectable virus in the airways or lungs (Fig. 4D, 4E ). They had 172 few signs of lung inflammation with moderate levels on inflammatory cells in the airways and 173 virtually none in the lung tissue (Fig. 4g) , and only baseline levels of all pro-inflammatory 174 cytokines in the airways, lung and serum (Fig. 4h, S2) . Importantly, combination of the spike 175 protein and alum with BCG did not alter the protective efficacy of the BCG vaccine against 176 aerosol M. tuberculosis in mice (Fig 4i) . as two weeks post-BCG:CoVac vaccination (Fig. 3) , we sought to determine if responses could 187 be further augmented by boosting with a prototype subunit vaccine (Alum SpK ) ( Fig. 5a ). At 7 188 days post-boost (day 28), IgG2c titres in plasma from mice primed either with BCG SpK or 189 BCG:CoVac were increased and remained elevated up to day 42 (Fig. 5b) . Corresponding 190 augmentation of NAbs was also seen in these boosted groups, with significantly elevated 191 responses in BCG:CoVac primed mice boosted with Alum SpK (Fig. 5c ). Boosting Alum SpK 192 vaccination with a second dose led to a greater than 10-fold increase in NAbs in boosted mice; 193 however, responses were significantly higher in those with the BCG:CoVac-prime, Alum boost combination (Fig. 5c) were approximately 10-fold less than those following the BCG:CoVac prime, Alum SpK 199 combination (Fig. 5e) . 200

Taken together, these data indicate that the antigen-specific immunity imparted by 201 BCG:CoVac can be further enhanced by heterologous boosting with a second SARS-CoV-2 202 vaccine, with this vaccination regime able to induce antibodies that can neutralize key VOCs. 203

Global vaccine access and distribution to low-and middle-income countries is critical for the 206 control of the COVID-19 pandemic. Vaccines must offer effective protective immunity yet 207 should be cheap to manufacture and have feasible cold chain management requirements. This 208 study describes a COVID-19 vaccine formulation, BCG:CoVac, that when delivered as a single 209 dose induces potent SARS-CoV-2 specific immunity in mice, particularly the generation of 210 high-titre, anti-viral neutralizing antibodies. Encouragingly, the level of immune responses 211 observed (particularly the generation of neutralizing antibodies) is equivalent to or exceeds 212 immunity elicited by approved COVID-19 vaccines, when these candidate vaccines were tested 213 in the murine model 24-26 . BCG:CoVac may have the additional advantage of inducing 214 protection against other respiratory infections for which BCG is known to induce some level 215 of protective immunity, including future pandemic viruses 11 . In addition, the possibility that 216 prior BCG exposure may impart protection against severe COVID-19 27 , which is currently 217 under evaluation through numerous randomised control studies 9 , raises the possibility that a 218 BCG-based vaccine could afford protection against SARS-CoV-2 escape mutants or new 219 pandemic coronavirus that may emerge. Indeed our data demonstrate that BCG:CoVac can 220 neutralize two of the key VOCs that are circulating globally, namely B.1.1.7 and B.1351. 221 BCG:CoVac could also provide additional benefit in countries where BCG is part of childhood 222 immunization programs for the control of TB, based on recent findings that repeat BCG 223 vaccination significantly reduced rates of M. tuberculosis infection 28 . 224

An advantage of our vaccine approach is the use of alum to potentiate immune responses, 225 particularly the generation of NAbs after vaccination. Alum is a low cost, globally accessible 226 vaccine adjuvant with an excellent safety record in humans 19 . The relative paucity of IFN-g-227 secreting T cells observed after Alum SpK vaccination corresponds with that previously seen 228 with alum-precipitated vaccines using the spike protein 29 and is consistent with the known 229 preferential priming of Th2-type immunity by alum-based adjuvants 30 to inactivate the reaction and then centrifuged again. Cell pellets were resuspended in 160 µL 382 HANKS solution and enumerated using a haemocytometer (Sigma-Aldrich, USA). Multi-lobe 383 lungs were collected and cut into equal thirds, before snap freezing on dry ice. Lung 384 homogenates were prepared fresh, with multi-lobe lungs placed into a gentleMACS C-tube 385 (Miltenyi Biotec, Australia) containing 2 mL HANKS solution. Tissue was homogenised using 386 a gentleMACS tissue homogeniser, after which homogenates were centrifuged (300 g, 7 min) 387 to pellet cells, followed by collection of supernatants for plaque assays and cytokine/chemokine 388 measurements. The single lobe lung was perfused with 0.9% NaCl solution via the heart, 389 followed by inflation with 0.5 mL 10% neutral buffered formalin through the trachea, and 390 placed into a tube containing 10% neutral buffered formalin. Following fixation for at least 2 391 weeks, single lobes were transported to a PC2 facility where they were paraffin-embedded, 392 sections cut to 3 µm thickness using a Leica microtome (Leica, Germany) and then stained 393 Briefly, a standard curve for each analyte was generated using a known standard supplied with 413 each CBA Flex kit. For each sample, 10 µL was added to a well in a 96-well plate, followed 414 by incubation with 1 µL of capture bead for each analyte (1 hr, RT, in the dark). Following 415 capture, 1 µL of detection bead for each analyte was added to each well, followed by incubation 416 

Measuring of IgG2c 479 isotype instead of IgG2a in immunized C57BL/6 mice with Plasmodium vivax TRAP as a 480 subunit vaccine candidate in order to correct interpretation of Th1 versus Th2 immune 481 response

An Effective COVID-19 Vaccine Needs to

The generation of T-cell memory to protect against 485 tuberculosis

SARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific Germinal 487

Neutralizing antibody levels are highly predictive of immune 490 protection from symptomatic SARS-CoV-2 infection

Rapid Generation of Neutralizing Antibody Responses in COVID-19

COVID-19 vaccines: neutralizing 494 antibodies and the alum advantage

Commission Statement on the occasion of the 75th session of the UN General Assembly

Animal and translational models of SARS-CoV-2 infection and 499 COVID-19