key: cord-0287295-hxrv2z83
authors: Raheja, Harsha; Das, Soma; Banerjee, Anindita; Dikshaya, P; Deepika, C; Mukhopadhyay, Debanjan; Ramachandra, Subbaraya G; Das, Saumitra
title: RG203KR mutations in SARS-CoV-2 Nucleocapsid: Assessing the impact using Virus-like particle model system
date: 2022-01-03
journal: bioRxiv
DOI: 10.1101/2022.01.02.473343
sha: 3f4d57fd42ecd32ac974f47b5869da5e8f0a866e
doc_id: 287295
cord_uid: hxrv2z83

The emergence and evolution of SARS-CoV-2 is characterized by the occurrence of diverse sets of mutations that affect virus characteristics, including transmissibility and antigenicity. Recent studies have focused mostly on Spike protein mutations; however, SARS-CoV-2 variants of interest (VoI) or concern (VoC) contain significant mutations in the nucleocapsid protein as well. To study the relevance of the mutations at the virion level, recombinant baculovirus expression system based VLPs were generated for the prototype Wuhan sequence along with Spike mutants like D614G, G1124V and the significant RG203KR mutation in Nucleocapsid. All the four structural proteins assembled in a particle wherein the morphology and size of the particle confirmed by TEM closely resembles the native virion. The VLP harbouring RG203KR mutations in nucleocapsid exhibited augmentation of humoral immune responses and enhanced neutralization by the immunized mice sera. Results demonstrate a non-infectious platform to quickly assess the implication of mutations in structural proteins of the emerging variant.

COVID-19 has been one of the leading causes of death globally since its emergence in 25 December 2019. The coronavirus, SARS-CoV-2, has been identified as the causative agent, 26 and it has a 30 kb single stranded genome encoding 4 structural and 16 non-structural proteins 27 (1). During infection, SARS-CoV-2 virus enters the cells through the ACE2 receptor (present 28 on the epithelial cells lining the respiratory tract), which is recognized specifically by the spike 29 protein SARS-CoV-2 virus (2). Along with the spike protein (S), Envelope (E) and Membrane immune response thereby generating specific antibodies against them (3, 4). Nucleocapsid has 33 been shown to be highly immunogenic and a promising vaccine target in SARS-CoV infection 34 as well (5, 6). New virus variants with mutations in these proteins are emerging continuously, 35 with increased transmissibility and severity. It is of utmost importance to understand the 36 molecular basis and effects of these mutations for an effective therapeutic and vaccine 37 development. However, it is challenging to study them because of Biosafety level 3(BSL-3) 38 requirement. We have designed Virus-like particle (VLP), which is composed of all the 39 structural proteins that form non-infectious virus-like particles but generate immune responses 40 similar to infectious virus particles enabling the study of mutation of all the structural proteins 41 in a more physiologically relevant system. The VLP has been produced using Baculovirus 42 mediated gene expression because of its advantages over Adenovirus and lentivirus systems 43 (7, 8) . The mutations, D614G and G1124V within spike and RG203KR within Nucleocapsid 44 revealed plausible structural implications as depicted through previous studies (9, 10). D614G 45 predominantly circulated worldwide and is presently incorporated into the backbone of all 46 emerging strains (VoCs and VoIs). Clinical evidence has revealed to increase viral replication 47 in the upper respiratory tract by augmenting infectivity and virion stability (11). G1124V is 48 one of the major mutations on CD8 T cell epitopes in S protein, which might have significant 49 implications in context to immunogenicity.

The R203K and G204R mutations in Nucleocapsid were first identified in the A2a lineage 51 within China and subsequently have spread within other lineages across Western Europe, UK, 52 and then to the US and other parts of the world through a number of VoCs and VoIs, viz., the 53 Alpha, Gamma, Lambda and now in the most underscored VoC, the Omicron. This RG203KR 54 mutation has been shown to enhance the infectivity, fitness and virulence (12) (13) (14) . Recently, a 55 different VLP approach has also been used to study the effect of Nucleocapsid mutations on 56 transmissibility of the virus (15). However, the impact on immunogenicity remains to be 57 studied. Here, we have incorporated these mutations to study their impact using VLP as a 58 platform. To assess the physiological binding of VLPs to the ACE-2 receptor, Vero E6 cells were used.

The VLP was fluorescence-labelled in vitro with Alexa Fluor 488 and its binding and were injected in mice and sera collected as mentioned ( Fig. 2A) . We first checked the VLP tolerance by injecting a high dose (100 ug) in 6 weeks old Balb/c mice. We took 4 groups of 89 mice, one for each VLP and one for vehicle control (PBS), with 6 mice in each group. The 90 mice were monitored for 4 weeks for appearance of any toxic symptoms. All the mice survived 91 with no effect on increase in body weight ( Fig 2B) . Additionally, administration of purified 92 VLPs did not affect the histology of mice liver, kidney, heart and lungs as observed by 93 histopathology (data not shown).

Humoral response generated against the WT-VLP was quantified using ELISA and all the three 95 VLP injected sera exhibited significant response as compared to vehicle control. Grossly, 96 immunization with S mut-VLP elicited a higher response after the booster dose in contrast to 97 WT or even S+N mut-VLP. (Fig. 2C ). Humoral immunity in the form of IgM (first class of 98 antibodies) and IgG (antibody produced after class switching) response was measured against The highest titre sera obtained upon immunization with VLPs was further used to check the 118 neutralisation of labelled VLP binding to Vero cells. We observed more than 50% VLP 119 neutralisation in the presence of 1:2 dilution of sera from mice immunized with all the 3 VLPs 120 (Fig. 2H) . Notably, the efficiency of neutralisation of S mut and S+N mut-VLP was higher than 121 the WT-VLP, pointing towards the accessibility of RBD in these VLPs for antibody 122 neutralisation, as observed previously.

Taken together, we provide a comprehensive report of the impact of RG203KR mutation in 124 nucleocapsid, on the immunogenicity and neutralisation efficiency using a model which can be 125 easily manipulated and exploited to study the emerging SARS-CoV-2 mutations in a system 126 closely resembling the virus while being non-infectious. It can be used to study the immune Transmission Electron microscopy (Immunogold labelling and negative staining): 153 The purified VLP was diluted in PBS, fixed with 4% paraformaldehyde and spotted onto 400 154 mesh carbon-coated copper grids for 10 min. It was then blocked using 1% BSA for 10 min, 155 which was followed by incubation with primary antibody against SARS-CoV-2 S protein (Cat.

No.-40592-R001) for 30 min. Thereafter, PBS wash was done 3-5 times and the grid was 157 incubated with gold conjugated anti-rabbit secondary antibody for 15 mins. After 7-8 PBS 158 washes, 1% glutaraldehyde was added onto the grid for 5 min to stabilise the immunostaining. 

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