key: cord-0331076-pbo0fmfw
authors: Counoupas, Claudio; Pino, Paco; Stella, Alberto O.; Ashley, Caroline; Lukeman, Hannah; Bhattacharyya, Nayan D.; Tada, Takuya; Anchisi, Stephanie; Metayer, Charles; Martinis, Jacopo; Aggarwal, Anupriya; Dcosta, Belinda M.; Kint, Joeri; Wurm, Maria J; Landau, Nathaniel R.; Steain, Megan; Turville, Stuart G; Wurm, Florian M; David, Sunil A.; Triccas, James A.
title: Neutralising antibodies against the SARS-CoV-2 Delta variant induced by Alhydroxyquim-II-adjuvanted trimeric spike antigens
date: 2021-08-19
journal: bioRxiv
DOI: 10.1101/2021.08.18.456891
sha: 8d6c886343be513733584b71908753144f31e14f
doc_id: 331076
cord_uid: pbo0fmfw

Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained and cross-variant SARS-CoV-2 neutralising antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a TLR7/8 small-molecule agonist chemisorbed on aluminium hydroxide. Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titre NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralising against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titres against multiple SARS-CoV-2 variants; notably high titres against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern.

40 COVID-19 vaccines have had a remarkable impact on controlling the pandemic in high and 41 middle-income countries. However, global access to affordable COVID-19 vaccines remains 42 a critical issue 1 . Neutralising antibodies (NAbs) are considered the key determinant of SARS-43

CoV-2 protective immunity 2,3 , yet in both natural infection and vaccination the levels of NAbs 44 decay over time 4, 5 . This issue is compounded by the emergence of SARS-CoV-2 variants 45 circulating that show partial resistance to current vaccines 6,7 , highlighting the need for next-46 generation vaccines that display strong and persistent immunity. 47 5 is a similar reduction as seen for other COVID-19 vaccines ( Fig. 2A) 7 . NAb titres using plasma 90 from SpK Alum -vaccinated mice were reduced to the limit of detection against Beta (Fig 2B) . 91

Cross-species neutralisation of VOCs was apparent after immunization of rabbits (Fig. 2C ) or 92 horses ( Fig.2D) with CoVac-II. NAb titres were maintained against the Alpha variant in both 93 species compared to ancestral virus, and high titres against Beta in rabbits (4.7-fold reduction 94 compared to ancestral) and horses (2.7-fold). Thus AHQ-II can adjuvant vaccine 95 immunogenicity across multiple animal models, adding to its already proven immunogenicity 96 in humans as part of the Covaxin vaccine 11 . 97 98 Although CoVac-II immunisation affords some level of cross-neutralisation against the Beta 99 variant, vaccines currently in use display reduced efficacy against this variant when assessed 100 in placebo-controlled or test-negative control trials 8,15,16 . We therefore used our established 101 systems for rapid and high-level production of trimeric viral antigens 10,17 to produce the Beta 102 spike antigen. Mice vaccinated with CoVac-II eight months earlier (or control groups) were 103 boosted with a single dose of Beta spike formulated in AHQ (CoVac351). Two weeks later, 104 the ability of their plasma to neutralize Beta spike-pseudotyped virus was determined 18 . The 105 increase of NAbs in response to this booster injection was greatest in mice previously 106 vaccinated with SpK Alum or CoVac-II (approximate increase of 16-fold compared to pre-boost 107 levels), however responses were maximal in the CoVac-II prime, CoVac351 boosted group 108 ( Fig. 2E ). High numbers of spike-specific, multifunctional Th1 CD4 + cells were observed in 109 boosting with CoVac351 resulted in enhanced NAb titres against all VOCs, with greatest 115 increase seen against the Beta variant ( Fig 2F) . Notably, neutralisation titres against the Delta 116 variant were high (>10 4 ) with only a small reduction in titre (approximately 2.2-fold) compared 117 to 'wild-type' virus. Of note, these NAb titres are at least 1 order of magnitude higher than the 118 average human convalescent response, which we have previously assessed with similar 119 methodology, and are comparable with that of 'elite' neutralisers 4 . 120 121

In conclusion, the CoVac-II subunit vaccine we described in this report demonstrates 122 remarkable longevity of immune responses (no decay in NAbs up to 8 months post-vaccination 123 in mice) and is highly immunogenic in multiple animal models, including rabbits and horses. 124

The waning of immunity observed in convalescent patients 4 and with current vaccines, coupled 125 with low NAb titres correlating with breakthrough infections 19 , suggests that maintenance of 126 humoral immunity will be critical to ensure prolonged vaccine-induced protection against 127 disease. NAbs developed in all immunised species are able to effectively neutralise SARS-128

CoV-2 variants of concern, which can be augmented by boosting with variant-specific spike 129 vaccines. CoVac-II-immunity compares favourably with other vaccines tested in the same 130 models, that have subsequently shown high-levels of protection in humans 13,20,21 . The excellent 131 safety profile and immunogenicity demonstrated by a AHQ-II-adjuvanted inactivated SARS-132

CoV-2 vaccine 11 , coupled with our ability to manufacture multiple, high quality antigens at 133 scale 10 , suggests that AHQ-II/spike protein combinations could constitute safe, affordable and 134 mass-manufacturable COVID-19 vaccines for global distribution. 135

Female C57BL/6 mice (6-8 weeks of age) were purchased from Australian BioResources 139 (Moss Vale, Australia), and housed at the Centenary Institute in specific pathogen-free 140 conditions. All mouse experiments were performed according to ethical guidelines as set out 141 by the Sydney Local Health District (SLHD) Animal Ethics and Welfare Committee. Table 1) . 173

Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm TM kit according to 174 the manufacturer's protocol and intracellular staining was performed to detect cytokines IFN-175 g, IL-2, TNF, IL-17 (see Supplementary Table 1 

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