key: cord-0291254-cuihpmrn
authors: Dubé, Charlotte; Paris-Robidas, Sarah; Andreani, Guadalupe; Gutzeit, Cindy; D'Aoust, Marc-André; Ward, Brian J.; Trépanier, Sonia
title: Broad Neutralization against SARS-CoV-2 Variants Induced by Ancestral and B.1.351 AS03-Adjuvanted Recombinant Plant-Derived Virus-Like Particle Vaccines
date: 2022-05-27
journal: nan
DOI: 10.1016/j.vaccine.2022.05.046
sha: 9b7925a42f61f301275cdf3f12dfd413719b8866
doc_id: 291254
cord_uid: cuihpmrn

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection resulting in the coronavirus disease 2019 (COVID-19) has afflicted hundreds of millions of people in a worldwide pandemic. Several safe and effective COVID-19 vaccines are now available. However, the rapid emergence of variants and risk of viral escape from vaccine-induced immunity emphasize the need to develop broadly protective vaccines. A recombinant plant-derived virus-like particle vaccine for the ancestral COVID-19 (CoVLP) recently authorized by Canadian Health Authorities and a modified CoVLP.B1351 targeting the B.1.351 variant (both formulated with the adjuvant AS03) were assessed in homologous and heterologous prime-boost regimen in mice. Both strategies induced strong and broadly cross-reactive neutralizing antibody (NAb) responses against several Variants of Concern (VOCs), including B.1.351/Beta, B.1.1.7/Alpha, P.1/Gamma, B.1.617.2/Delta and B.1.1.529/Omicron strains. The NAb response was robust with both primary vaccination strategies and tended to be higher for almost all VOCs following the heterologous prime-boost regimen.

Since the declaration of a pandemic situation caused by the SARS-CoV-2 by the World Health Organisation (WHO), over 410 million cases have been reported and more than 5.8 million people have died from COVID-19 (WHO Coronavirus Disease (COVID- 19) Dashboard, https://covid19.who.int/, 2021). The rapid development and approval of vaccines with efficacy up to 95% led to hope in mid-2021 that the worst of the pandemic was over [1] [2] [3] [4] . However, the total number of COVID-19 cases is still growing rapidly worldwide with almost 300 000 reported deaths in just the last month, mostly attributable to highly transmissible SARS-CoV-2 Variants of Concern (VOCs). The most worrisome variants are those with mutations in the Spike (S) protein that not only enhance transmissibility but also increase virulence and evasion of vaccine-induced immunity [5] [6] [7] [8] [9] [10] or resistance to neutralization by monoclonal antibodies [8, 9, 11] . The S protein plays a crucial role in SARS-CoV-2 infection through the interaction of its receptor binding domain (RBD) with the angiotensin-converting enzyme 2 (ACE2) receptor on host respiratory epithelial cells [12] [13] [14] . All of the currently approved vaccines target the S protein of the ancestral strain of SARS-CoV-2 identified in Wuhan and a growing number of reports demonstrate that their efficacy against mainly the B.1.351 and the B.1.617.2 variants is reduced [15] [16] [17] [18] .Medicago has developed a SARS-CoV-2 vaccine using a platform technology based on transient expression of recombinant proteins in non-transgenic Nicotiana benthamiana plants and a disarmed Agrobacterium tumefaciens as a transfer vector to move targeted DNA constructs into the plant cells [19] . The S protein trimers displayed on the surface of the plant-derived coronavirus-like particles (CoVLP) are in a stabilized, prefusion conformation that resemble native structures on Page 4/28 wild-type SARS-CoV-2 virions. Plant-based VLP vaccines are an emerging production platform that has many potential advantages such as proper eukaryotic protein modification and assembly, low risk of contamination with adventitious agents, scalability, and rapid production speed [20] . Currently, several plant-based VLP vaccine candidates against pathogens such as Hepatitis B virus [21] , Rabies virus [22] , Influenza virus [23] and Norwalk virus [24] are under clinical development. At the time of writing, only two plant-based VLP vaccine candidates against SARS-CoV-2 have reached the clinical stage; Medicago's CoVLP has completed its primary vaccine efficacy analyses in Phase 3 (NCT04636697) and has recently been authorized by Canadian Health Authorities [25] and Kentucky Bioprocessing-201 is in Phase 1/2 (NCT04473690).

Herein, we present the preclinical evaluation of a CoVLP candidate targeting the B. Page 5/28

The full-length S glycoprotein of SARS-CoV-2 from the GISAID database 

The PNA was performed by Nexelis (Laval, Quebec, Canada) using a pseudovirus based on SARS-CoV-2 ancestral Wuhan strain (reference MN908947) as previously described [27] . Analyses were performed in duplicate and included appropriate controls. The assay was qualified for the ancestral pseudovirus strain. Cross-reactivity was evaluated using cell growth media at a starting dilution of 1/25 or 1/250, followed by a serial dilution (2fold dilutions, 5 times). A previously pre-determined concentration of pseudovirus was then added to diluted sera samples and pre-incubated for 1 hour at 37°C with CO 2 . The mixture was then added to pre-seeded confluent Vero E6 cells expressing the ACE2 receptor (ATCC CRL-1586) and incubated for 18-24 hours at 37°C with 5% CO 2 .

Following incubation and removal of media, ONE-Glo EX Luciferase Assay Substrate (Promega, Madison, WI) was added to cells and incubated for 3 minutes at room temperature with shaking. Luminescence was measured using a SpectraMax i3x microplate reader (Molecular Devices, San Jose, CA). A titration curve was generated based on a 4-parameter logistic regression (4PL) using Microsoft Excel. The NAb titer was defined as the reciprocal of the sample dilution for which the luminescence was equal to a pre-determined cut-point value corresponding to 50 % neutralization.

Responders were considered positive if the NAb titer was ≥ 25. Neutralization antibody results presented in the current study were obtained using pseudotyped SARS-CoV-2 virus. Note that results obtained with pseudovirus neutralization assay generally correlates with live virus based microneutralization assay [28] .

The descriptive statistics and statistical comparisons were performed using GraphPad Prism software (Version 8.4.2; GraphPad Prism Software, La Jolla, CA, USA). The geometric mean titers (GMT) of NAb titers with 95 % confidence intervals (CI) and percentage of positive responders were calculated for each group of mice. A titer value of 12.5 was attributed to titers lower than the minimum required dilution (MRD) (i.e., 1/25).

Statistical comparisons to evaluate differences between groups were performed using either a one-way ANOVA followed by a Tukey post hoc test, or a two-way ANOVA followed by a Bonferroni post hoc test on log 10 -transformed antibody titers. Wilcoxon matched-pairs signed rank was used to assess differences between the various SARS-CoV-2 pseudovirus strains. The threshold for statistical significance was set to p < 0.05.

In this study, mice were immunized following either a homologous or heterologous prime-boost regimen with AS03-adjuvanted CoVLP and/or CoVLP.B1351 (Figure 1 

The heterologous, AS03-adjuvanted CoVLP-CoVLP. 

Overall, no safety concerns were raised following homologous prime-boost strategies or the heterologous strategy. The post-immunization variations observed for body weight and food consumption were transient and/or within the normal variations ( Figures S1 and   S2 ). An unexpected increase in food consumption was observed in the CoVLP + AS03 group between Days 7-14 that was likely attributable to eating-like behavior (i.e. stashing of food pellets at the bottom of the cage) in a small number of the animals in this group. Transient signs of discomfort (Table S1 ) and inflammation at the dosing sites (edema and erythema) were reported in all treated groups following the prime ( Figure   S3 ). All observations generally subsided within 10-14 days and were no longer seen prior to the second administration. After the second administration, no signs of reactogenicity or discomfort were reported at the injection site. No macroscopic anomalies were reported following euthanasia and collection of organs and tissues.

The first anti-COVID-19 vaccines were approved for emergency use within a year of the start of the pandemic with reported efficacies ranging from 50-95% [29] . Simultaneously however, multiple viral variants have emerged in different geographic regions with varied transmissibility, virulence and resistance to vaccine-induced immunity [30] [31] [32] [33] [34] [35] . Many parts of the world have experienced rapid replacement of the ancestral Wuhan-like strain with one or a sequence of these variants of concern "VOCs". The different waves of variants has complicated diagnostic efforts in some cases [8, 9, 36] and generally frustrated efforts to control the spread and impact of the pandemic [ [42, 43] and it is estimated that the transmission rate of the recent B.1.1.529 variant is at least 4-times higher than the ancestral strain [44] . Several lines of evidence including animal models and epidemiological observations suggest that this increased transmissibility is related to Spike protein mutations [45, 46] such as D614G [47, 48] , P681R [49] or K417N/E484K/N501Y [50] . These mutations have significant functional effects related to transmissibility such as accelerated cell-to-cell spread [49] and the generation of higher virus loads in the upper airways when compared to the B.1.1.7 strain [42] . Although the relationship between different mutations and disease severity is not yet fully understood [51] , recent evidence suggests that the B. [5, 11, 18, 58, 59] . In several randomized controlled trials with different vaccines, efficacy against the B.1.351 variant was observed to fall by 33-84% [15] [16] [17] 60] .

Although most of the deployed vaccines continue to have good efficacy against severe disease induced by most of the VOCs identified to date [15, 61] and there is evidence of convergent evolution [62, 63] , it is unlikely that SARS-CoV-2 has fully exhausted its genetic repertoire. These observations highlight the need both to evaluate the ability of vaccines already deployed or in advanced development to neutralize the VOCs and to develop next generation vaccines with broader cross-reactivity. In this study, the cross- [43, 56, 59] and studies in non-human primates immunized with ancestral SARS-CoV-2 vaccines [66, 67] . In the current study, the decrease in neutralizing activity was less pronounced following the administration of the candidate B.1.351 vaccine compared to the vaccine based on the ancestral strain, possibly due to the closer phylogenic relationship between B.1.351 and the Omicron variants [68] .

Despite these promising data, it is possible that one or more VOCs will eventually emerge that is/are no longer effectively neutralized by vaccine-induced immunity. It is in this context that Medicago and others have chosen to develop next-generation vaccine candidates targeting the B.1.351 strain since this strain is one of the most antigenically distant VOC to emerge to date. The B.1.351 variant has consistently proved to be difficult to neutralize in vitro [69, 70] and has caused large decrements in vaccine efficacy in several randomized controlled trials [15] [16] [17] 61 ]. In the current study, animals that received two doses of either AS03-adjuvanted CoVLP or CoVLP.B1351 mounted neutralizing antibody responses that were comparable for both homologous and heterologous strains while reports for other candidate B.1.351 vaccines in mice have shown either strong homologous (ie: B.1.351-specific) responses only [71] or the requirement for three doses to achieve high levels of neutralizing antibodies [64] . The pattern of the neutralizing antibody response was consistent across multiple VOCs in the current study with the CoVLP.B1351 candidate generally eliciting higher titers than the ancestral CoVLP and this difference reached significance for the P.1 (2.3x) and the B.1.1.529 (6.8x) strains. Although the level of cross-neutralization in the animals that received CoVLP.1351 was lower for the B.1.1.7 (-1.6x) and B.1.617.2 (-4.0x) variants compared to the homologous response, such differences are expected given the genetic and antigen 'distance' between these VOCs [43] . Furthermore, while these relative decreases were observed, the absolute titers of cross-reactive antibodies induced by two doses of CoVLP.B1351 with AS03 against the VOCs tested was still substantial. These findings are consistent with observations of others [64, 71, 72] and suggest that vaccines targeting the original Wuhan-like strain may be eventually become suboptimal in the next stages of the pandemic, opening the door to less conventional vaccination approaches including heterologous prime-boost strategies.

Concern over the ability of any single S protein antigen to elicit a broad enough response to neutralize all of the known and possibly future VOCs prompted us to evaluate the possible benefits of a heterologous prime-boost strategy with the Wuhan-like CoVLP as the prime and CoVLP.B1351 as the boost; both adjuvanted with AS03. Heterologous vaccination strategies that use two distinct platforms and/or deliver two slightly different antigens have shown considerable promises for a wide range of viral pathogens that rapidly mutate such as HIV [73] , hepatitis C virus [74] or influenza to both broaden the immune response and focus the response on conserved epitopes [75] . This approach was largely confirmed in the current study since the neutralizing antibody titers were consistently higher in the animals that had received the AS03-adjuvanted CoVLP-CoVLP.B1351 regimen, reaching statistical significance over the AS03-adjuvanted CoVLP-CoVLP regimen for B.1.351 and P.1 strains and over the AS03-adjuvanted CoVLP.B1351-CoVLP.B1351 regimen for the ancestral strain. It is not currently known if these differences between high and very high neutralizing antibody responses will have any clinical significance. However, induction of very high initial titers is likely desirable since it is well-documented that antibody titers wane substantially with time after both natural disease and vaccination [76] . These observations are similar to the results recently released by others [72, 77] [Pfizer, Novavax] but distinct from those reported by Moderna [71] in that no evidence of original antigenic sin was noted [78] . Since these animals only received two doses, it is currently unknown how humoral response against VOCs would be influenced by a third (booster) dose but others have reported very high and cross-protective neutralizing antibody responses both in animals [64, 71] and human trials [65, 79, 80] after this additional dose.

Finally, it is worth noting that these observations focus entirely on vaccine-induced antibody responses and particularly on the induction of antibodies capable of neutralizing SARS-CoV-2 variants in vitro. Although many consider neutralizing antibody levels to be a good candidate for a correlate of protection [81] , this is a fairly limited evaluation of vaccine-induced immunity and it is very likely that non-neutralizing but functional antibodies and cellular responses also contribute to vaccine-induced protection [82] . Data from a large non-human primate study [83] as well as ongoing clinical trials [27, 65, 84] demonstrate that AS03-adjuvanted CoVLP stimulates multiple arms of the adaptive response to SARS-CoV-2. Results from Medicago's ongoing pivotal Phase 3 efficacy study [25] 

Page 18 /28 The study was sponsored by Medicago Inc. The authors would like to acknowledge Philippe Boutet, Margherita Coccia, Marie-Ange Demoitié, Ulrike Krause and Eric Destexhe from GSK for critical review of the manuscript. The authors also wish to acknowledge all the Medicago employees and their contractors (ITR Laboratories Canada Inc and Nexelis) for their exceptional dedication and professionalism.

All authors contributed significantly to the submitted work. CD, SPR, GA, CG, BJW and ST contributed to design and execution of the study as well as analyses and presentation of the data. All authors contributed to critical review of the data and the writing of the manuscript. All Medicago authors had full access to the data. (formulated with AS03 adjuvant) or control. Food consumption was measured weekly throughout the study period on all animals (8/group, excepted for control 5/group). Results are reported as mean weekly food consumption (± SD) per group. Statistical comparisons with the control were performed for each 7-day study period using a non-parametric Kruskal-Wallis analysis followed by a Dunnett's test on ranks.

Significant differences were annotated as * p < 0.05, ** p < 0.01 and *** p < 0.001. Aberrant measurement was reported for CoVLP + AS03 between Days 7-14 and could be explained be eating like behavior reported in animals. Therefore, only results after the first immunization are shown. Data are presented as mean Draize scoring ± SD.

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☒ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

CD, SPR, GA, MAD, BJW and ST are either employees of Medicago Inc or receive salary support from Medicago Inc. CG is an employee of the GSK group of companies and reports ownership of GSK shares.

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NAb titers were measured against SARS-CoV-2 pseudoparticles in serum samples using a cell-based PNA targeting the ancestral or B.1.351 strains. Half of the minimum required dilution (MRD) of the method was assigned to non-responders (i.e. 12.5). (A) GMT with 95 % CI measured 21 days after the 1 st immunization (Day 21) and 14 days after the 2 nd immunization (Day 35). Statistical comparisons were performed using a two-way ANOVA followed by a Bonferroni post hoc test on log 10 -transformed NAb titers. (B-C) Results from individual mouse serum samples (n=8 per antigen) are represented as dots on each figure with lines connecting ancestral of B

Cross-Reactive Neutralization against the ancestral, Beta (B.1.351) Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529) strains Following Homologous or Heterologous Prime-Boost Regimen

NAb titers were measured against SARS-CoV-2 pseudoparticles in serum samples using a cell-based PNA targeting the ancestral

Day 35) for the (A) ancestral, (B) B.1.351, (C) B.1.1.7, (D) P.1, (E) B.1.617.2 or (F) B.1.1.529 strains. Statistical comparisons between the CoVLP-treated groups were performed using a One-way ANOVA followed by a Tukey post hoc test (Day 35) on log 10 -transformed NAb titers. Significant differences are indicated with p-values on the graphs

BALB/c mice (n=8) were immunized IM on Days 0 with 3.75 µg CoVLP and 21 with 3.75 µg of

NAb titers were measured against SARS-CoV-2 pseudoparticles in serum samples using a cell-based PNA targeting the ancestral, B.1.351, B.1.1.7, P.1, B.1.617.2 or B.1.1.529 strains. Half of the minimum required dilution (MRD) of the method was assigned to non-responders (i.e. 12.5). Results from individual mouse sera (n=8 per antigen) are represented as dots on each figure with lines connecting the ancestral or B.1.351 variant to the B.1.351, B.1.1.7, P.1, B.1.617.2 or B.1.1.529 neutralization titers. Statistical comparisons were performed using Wilcoxon matched pairs signed rank test

Broad Neutralization against SARS-CoV-2 Variants Induced by Ancestral and B.1.351 AS03-Adjuvanted Recombinant Plant-Derived Virus-Like Particle Vaccines