Live attenuated virus vaccine protects against SARS-CoV-2 variants of concern B.1.1.7 (Alpha) and B.1.351 (Beta)

Description


INTRODUCTION
The devastation wrought by the coronavirus disease 2019 (COVID- 19) pandemic highlighted the urgent need for effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Vaccine development began immediately after the discovery of the virus and proceeded at an unprecedented pace and scale (1). Within less than a year from the onset of the pandemic, the concentrated efforts were crowned with the first vaccine authorizations (2,3). Shortly thereafter, vaccination campaigns began in many countries, focusing primarily on high-risk groups, such as the elderly and health care workers. Until now, mass vaccination campaigns confirmed that the licensed vaccines are highly effective in preventing symptomatic and asymptomatic SARS-CoV-2 infections, COVID-19-related hospitalizations, and deaths (4,5). To date, several vaccines have received emergency or full approval for use in many different countries (1,6), and additional vaccine candidates are in final phase 3 clinical trials (1,6). Although vaccine rollouts have been successful, primarily in many wealthy countries, vaccine supply remains scarce, and policymakers in most countries must prioritize vaccination programs to achieve the greatest public health benefit. To reduce the immense social and economic burden and ultimately stop the pandemic, it will be necessary to develop, manufacture, and administer billions of safe, effective, and affordable vaccine doses to a large proportion of the world's population, likely on a recurring basis.
SARS-CoV-2, positioned at the opposite end of the incipient arms race, is rapidly evolving (7)(8)(9). Benefiting from its global presence, the virus continues to adapt to its new host and to infectionor vaccine-induced immunity. During the course of the pandemic, a number of genetic variants have emerged (7)(8)(9). Variants that exhibit increased infectivity, cause greater morbidity and mortality, or have the ability to evade infection-or vaccine-induced immunity pose an increased threat to public health. The World Health Organization (WHO) and other national health agencies have independently established classification systems that categorize emerging variants as variants of interest (VOIs), variants under investigation (VUIs), or variants of concern (VOCs) based on their risk to public health (Table 1) (10)(11)(12)(13)(14). In addition, to simplify communication with the public, the WHO recommends that VOIs and VOCs should also be labeled using the letters of the Greek alphabet. As of 12 August 2021, viruses belonging to lineages B.1.1.7 (Alpha), B.1.351 (Beta), B.1.1.28.1 (Gamma), and B.1.617.2 (Delta) are classified by several health agencies as VOCs (Table 1). In countries where they emerged, these variants rapidly supplanted the preexisting variants and started to spread globally.
The B.1.1.7 variant, first detected in the United Kingdom in December 2020, is 50 to 100% more transmissible and possibly also more lethal than earlier variants but shows no tendency to evade immunity induced by infection or vaccination (9,15,16 (20).

RESULTS
We constructed a series of live attenuated SARS-CoV-2 vaccine candidates by large-scale recoding of the SARS-CoV-2 genome (21). The SARS-CoV-2 genome was modified by codon pair deoptimization (CPD), an approach that has resulted in rapid and efficient attenuation of a variety of RNA (22,23) and DNA viruses (24,25). CPD is based on the observation that some codon pairs occur in protein-coding sequences significantly less or more frequently than would be statistically expected (26). In CPD, viral genes are recoded to contain an increased number of codon pairs that are statistically underrepresented (suboptimal) in the host and are designed to lead to attenuation of the recoded viruses (22)(23)(24). In the process of CPD recoding, the positions of synonymous codons in the recoded sequence are exchanged in such a way that the frequency of underrepresented codon pairs increases. Because CPD exchanges only the positions of synonymous codons in recoded sequences, the recoded, live attenuated virus contains exactly the same antigens as the pathogenic parent. The introduced genetic changes reduce protein production from the recoded genes and also the replication fitness of the mutant virus (23). The conserved antigenic identity and the remaining replicative potential enable the recoded attenuated virus to fully engage the immune system of the host and provoke strong immune responses (22)(23)(24).
In our previous study, we have evaluated live attenuated virus candidates and have shown that some of them are strongly attenuated, induce strong immune responses, and protect Syrian hamsters from a challenge with ancestral wild-type (WT) SARS-CoV-2 (21). In that study, we also showed that the lead live attenuated virus candidate sCPD9 (Fig. 1A) is strongly attenuated in the Roborovski dwarf hamster (Phodopus roborovskii), which is highly susceptible to severe COVID-19-like disease (27). Despite the high susceptibility of this hamster species, sCPD9 did not cause clinical disease or considerable lung pathology (21).
The sCPD9 virus is also highly attenuated in vitro, where it grows to 100-fold lower titers than WT virus under normal conditions on Vero E6 cells (21). Nonetheless, it is possible to produce virus stocks with high titers (1 × 10 7 infectious virus particles/ml) using the procedure described in Materials and Methods. In the current study, we assessed immunogenicity and protective efficacy of the sCPD9 virus against the ancestral B.1 and two VOCs, B.1.1.7 and B.1.351.

Vaccination
Hamsters were randomly assigned to two groups of 12 and 30 animals. On day 0, 12 hamsters were mock-vaccinated and 30 hamsters were vaccinated with 1 × 10 5 focus-forming units (FFUs) of the sCPD9 virus by intranasal instillation. On day 21, hamsters were challenged with 1 × 10 5 plaque-forming units (PFUs) of the variants On average, sCPD9-vaccinated hamsters gained slightly less weight than mock-vaccinated hamsters in the 21 days after vaccination, but differences were not significant (Fig. 1B). No adverse reactions or clinical signs, such as forced breathing or apathy, that would indicate severe disease were observed in any of the mock-or sCPD9vaccinated hamsters. Attenuated sCPD9 virus replicated efficiently in the upper and lower respiratory tract of sCPD9-vaccinated hamsters on day 3 after vaccination ( Fig. 1C). In addition, we detected infectious viruses in the lungs of two-thirds of sCPD9-vaccinated animals on day 3 after vaccination ( Fig. 1C). Histopathological examination of lungs of sCPD9-vaccinated hamsters on day 3 after vaccination showed that sCPD9 is strongly attenuated, as only few mild inflammatory lesions could be detected in the lungs of vaccinated animals (21).

Challenge infection
Vaccination with the sCPD9 virus induced strong protective immunity against challenge with all three SARS-CoV-2 variants. None of the vaccinated hamsters showed clinical signs of disease, and none of the challenged groups lost weight after challenge (Fig. 1D). In contrast, hamsters of the mock-vaccinated group showed typical signs of severe COVID-19-like disease, began to lose weight on day 2 after challenge, and continued to lose weight until the end of the study (day 5 after challenge) (Fig. 1D).
Virological assessments of the challenge infection confirmed virus replication in the upper airways of infected animals on days 2, 3, and 5 after challenge (Fig. 1E). The detection of viral RNA in the upper respiratory tract of many vaccinated animals suggests that the virus remains capable of establishing local infection, regardless of the variant used for challenge. Nevertheless, vaccination drastically reduced the amount of viral genomic RNA in the lower respiratory tract of challenge-infected hamsters (Fig. 1F). Vaccinated hamsters had distinctly lower viral loads than control animals on all days examined. The viral load in the control group was on average one-to sixfold higher on day 2 and four-to fivefold higher on days 3 and 5 after challenge (Fig. 1F). As expected, variants replicated with unequal efficiency in the lungs of challenged hamsters. Hamsters infected with the ancestral SARS-CoV-2 variant B.1, which is antigenically identical with the sCPD9 virus, produced the lowest viral loads, whereas those infected with B.1.351 virus had the highest viral loads.
To further evaluate the immunity established by vaccination, we attempted to isolate the challenge virus from lungs of animals in cultured Vero E6 cells. This evaluation showed sterilizing immunity in the lower airways of all but one subject. While we failed to isolate B.1 and B.1.1.7 viruses, we detected infectious, replication-competent virus in a single hamster (one of three hamsters) in the B.1.351 group, but only shortly after challenge infection, on day 2 after challenge ( Fig. 1G).

Histopathology after challenge
To determine the protective capacity of vaccination with the sCPD9 virus against COVID-19-like disease caused by three different SARS-CoV-2 variants, the lungs of challenged hamsters were subjected to detailed histopathological examination (Fig. 2). The examination revealed that all sCPD9-vaccinated hamsters were fully protected against SARS-CoV-2-induced inflammatory damage to the lung, irrespective of the variant used for challenge infection (Fig. 2). In contrast, mock-vaccinated and B.1-infected hamsters developed severe lung pathology typical of experimental SARS-CoV-2 infection (27). These observations support the conclusion that vaccination with sCPD9 virus protected hamsters from major tissue damage and provided complete protection against COVID-19-like disease after challenge infection with three different SARS-CoV-2 variants.

Serum neutralization after challenge infection
We quantified the capacity of 17 hamster sera that were collected on days 2 and 3 after challenge to neutralize SARS-CoV-2 variants B.1, B. 1.1.7, B.1.351, B.1.128.1, and B.1.617.2 by a microneutralization assay (Fig. 3A). Sera from mock-vaccinated hamsters had no neutralization activity against all SARS-CoV-2 variants tested. Sera from sCPD9-vaccianted hamsters had strong neutralizing activity against the B.1 and B.1.1.7 variants, with geometric mean titers of 873 and 1129, respectively, but lower neutralizing activity against the B.1.351, B.1.128.1, or B.1.617.2 variants, with geometric mean titers of 243, 341, and 182, respectively (Fig. 3A). Challenge infection is expected to boost vaccine-induced immune responses and lead to production of antibodies that better neutralize antigens of the respective challenge SARS-CoV-2 variants. Because we collected sera shortly after the challenge, we did not expect to observe the induction of a variant-specific humoral response. Nevertheless, to account for possible challenge-induced effects, we also compared serum neutralization activity as a function of the challenge virus (Fig. 3B). This comparison showed that none of the different challenge viruses had any discernible effect on the neutralizing activity of the hamster sera. In other words, hamsters challenged with one variant, for example, B.1.351, did not produce a better humoral response against the same variant within 3 days after challenge. Furthermore, there was no significant difference in the neutralizing activity of sera collected on day 2 or 3 after challenge ( fig. S1).
Vaccination with sCPD9 induced high levels of neutralizing antibodies that were similarly effective against not only the ancestral B.1 variant, which is antigenically identical with the sCPD9 virus, but also the evolutionarily more distant variant B.1.1.7, which has 17 mutations that alter or delete amino acids (8 mutations in the spike protein) compared with the original virus. Hamster sera also effectively neutralized the B.1.351 variant, albeit with six-to ninefold reduced efficacy (Fig. 3C). The results are in good agreement with previous studies, which showed that B.1.351 markedly decreased the neutralization activity of convalescent plasma of individuals that were infected with early SARS-CoV-2 lineages (~9-to 15-fold reduction) (28-30) and sera from individuals who have been vaccinated with vaccines containing the original version of spike protein (~10-to 14-fold reduction) (30,31).
Moreover, sera from vaccinated hamsters collected on days 2 and 3 after challenge also efficiently neutralized SARS-CoV-2 variants B.1.128.1 and B.1.617.2 (Fig. 3, A to C). These two variants were not used for challenge infection in this study, but were still neutralized with similar efficacy (~5-to 8-fold reduction in comparison to B.1) as the B.1.351 variant (~6-fold reduction). This result confirms that vaccination with the vaccine candidate sCPD9 elicits broad and potent neutralizing antibody responses against a wide variety of SARS-CoV-2 variants. Our data also show that among the virus variants studied here, variant B.1.617.2 is the most resistant to neutralization with antibodies that were induced by vaccination with a virus that contained the ancestral form of the spike protein. Recent studies have yielded similar results, showing that sera from vaccinated individuals and convalescent sera from individuals previously infected with variants other than B.1.617.2 neutralized the B.1.617.2 variant with a three-to sixfold reduced efficacy (32,33).

DISCUSSION
Emerging variants are a constantly growing threat, because they not only dampen the efficacy of the licensed vaccines that target the original SARS-CoV-2 but also raise concerns that the virus may soon become resistant to current vaccines (34). Mutations in the spike protein have been shown to be primarily responsible for decreased neutralization of B.1.351 virus because they interfere with the efficient binding of neutralizing antibodies. Of particular importance is the E484K mutation in the receptor-binding domain of the spike protein, which prevents binding of the most potent neutralizing antibodies found in convalescent and postvaccination sera (35). This mutation is also present in B.   In the context of continuous SARS-CoV-2 evolution, our study yielded several important findings. We show that single-dose intranasal vaccination with a live attenuated virus vaccine candidate, sCPD9, induced complete protection against clinical disease and lung pathology caused by three different SARS-CoV-2 variants in a highly sensitive animal model. The challenge-infected hamsters did not develop any signs of disease and mounted strong neutralizing antibody responses. We show that B.1.351 virus, although markedly more resistant to neutralization by serum antibodies than B.1 or B.1.1.7 variants, was well controlled by vaccination with sCPD9. The fact that the lungs of B.1.351-challenged hamsters were completely protected from any damage suggests that vaccination with    Safe blood collection from dwarf hamsters before euthanasia does not yield sufficient serum to determine serum neutralization titers before challenge infection. The lack of access to the sera limits our ability to assess the antibody responses by the vaccine in the absence of infection. Excellent clinical protection, consistent with the absence of replication-competent virus in the lungs of challenged animals, and robust antibody responses shortly after challenge do, however, demonstrate the strong protective potential of this vaccine against different SARS-CoV-2 variants. In addition, vaccination with a live attenuated virus may offer better protection from (re)infection than vaccines that carry only a single antigen, because all SARS-CoV-2 proteins may trigger stronger cytotoxic T cell responses (38).

Ratio of neutralization titers
While other studies have shown that emerging variants threaten the success of vaccination and rekindle the pandemic by diminishing the efficacy of licensed vaccines, our study shows that a singledose intranasal vaccination with the lead live attenuated virus candidate sCPD9 induces immunity that provides excellent protection against currently circulating VOCs. It is conceivable that live attenuated virus vaccines, once fully developed, may offer a more practical solution on how to achieve protective immunity against such variants, instead of developing a specialized vaccine against each new dangerous variant that emerges. A live attenuated vaccine as described here may be particularly beneficial to boost and broaden immune responses after primary immunization with other vaccines. It remains to be determined to what extent the vaccination with the sCPD9 virus can protect against emerging VOCs in a nonhuman primate model or in humans.

Production of high-titer virus stocks of sCPD9
To produce high-titer virus stocks, confluent monolayers of Vero E6 cells grown in T-75 flasks were infected with sCPD9 virus at a multiplicity of infection of 1. Infected cells were cultured in 7 ml of cell culture medium and harvested at the onset of cytopathic effect on days 2 to 3 after infection. The virus stocks prepared in this manner typically contain approximately 1 × 10 7 infectious virus particles per milliliter of cell culture.

Virus titrations
The concentration of infectious viruses in virus stocks and in the lungs of infected animals was determined by titration on Vero E6 cells grown in 12-well plates. Stock titers of WT SARS-CoV-2 variants were determined by a plaque assay. Because sCPD9 virus does not produce readily visible plaques on Vero E6 cells, the titers of sCPD9 virus stocks were determined by a focus-forming assay. Briefly, Vero E6 cells were infected with 100 l of serial 10-fold dilutions of virus. After 2 hours of incubation, the viral inoculum was removed and cells were overlaid with Dulbecco's modified Eagle's medium (DMEM) containing 2.5% microcrystalline cellulose (Avicel, FMC BioPolymer). Seventy-two hours after infection, cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS). Cells were then incubated with a monoclonal mouse anti-SARS-CoV-2 nucleocapsid antibody (provided by S. Reiche, Friedrich Loeffler Institute, Riems, Germany) for 1 hour and then with goat anti-mouse immunoglobulin G-Alexa Fluor 568 secondary antibody (Thermo Fisher Scientific) for 45 min. To determine virus titers in the lung, 50 mg of lung tissue was first homogenized with a bead mill (Analytic Jena), the homogenate was serially diluted, and the virus titer was determined by focus-forming assay as described above.
After challenge infection of hamsters, virus titers in the lung of challenged animals were determined by plating 10-fold serial dilutions of 50 mg of lung homogenates onto Vero E6 cells. After incubation at 37°C for 2 hours, the homogenized tissue was aspirated, and cells were washed with PBS and overlaid with 2.5% microcrystalline cellulose (Avicel, FMC BioPolymer). Seventy-two hours after infection, cells were fixed with 4% formaldehyde and stained with crystal violet (0.75% aqueous solution), and plaques were counted.

Ethics statement
In vitro and animal work was done under biosafety conditions in the biosafety level-3 (BSL-3) facility at the Institut für Virologie, Freie Universität Berlin, Germany. All animal experiments were approved by the Landesamt für Gesundheit und Soziales in Berlin, Germany (permit number 0086/20) and performed in compliance with relevant institutional, national, and international guidelines for care and humane use of animal subjects.

Animal husbandry
The animal experiments were done in a certified BSL-3 facility. Fiveto seven-week-old Roborovski dwarf hamsters (P. roborovskii) were purchased through the German pet trade from a single breeding facility. They were housed in groups of six animals in individually ventilated cages. The animals had unrestricted access to food and water and were allowed to acclimate to the conditions for 7 days before infection. During both experiments, cage temperatures ranged from 22° to 24°C and relative humidity ranged from 40 to 55%.

Vaccination and challenge experiments
To assess the vaccine efficacy of the lead attenuated virus vaccine candidate sCPD9 in the Roborovski dwarf hamster, animals were randomly assigned into two groups, with 60% of the animals in each group being female. Twelve hamsters were mock-vaccinated with cell culture medium, and 30 hamsters were vaccinated with 1 × 10 5 FFU of the vaccine candidate sCPD9 virus in 20 l by intranasal instillation under anesthesia (40). Twenty-one days after vaccination, hamsters were challenge-infected with 1 × 10 5 PFUs of challenge virus in 20 l by intranasal instillation. Nine mock-vaccinated hamsters were challenged with the SARS-CoV-2 variant B.1, and nine sCPD9-vaccinated hamsters were challenged with either the SARS-CoV-2 variant B.1, B.1.1.7, or B.1.351. During the experiment, all hamsters were monitored twice daily for the clinical signs of disease and body weights were recorded. On day 3 after vaccination, and on days 2, 3, and 5 days after challenge (days 23, 24, and 26 of the experiment), three hamsters of each group were euthanized. Blood, tracheal swabs, and lungs were collected to determine virological and histological parameters of vaccination or infection. The left lungs were preserved in 4% formalin for subsequent histopathological detailed investigations.

Histopathology and in situ hybridization
For histopathology, the left lung lobe was carefully removed and immersion-fixed in buffered 4% formalin, pH 7.0, for 48 hours. Lungs were embedded in paraffin and cut at 2 m thickness. Sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) reaction followed by blinded microscopic evaluation by board-certified veterinary pathologist (A.D.G.) (41). H&E-and PAS-stained and in situ hybridization slides were analyzed and photographed using an Olympus BX41 microscope with the DP80 Microscope Digital Camera and the cellSens Imaging Software, version 1.18 (Olympus Corporation). For overviews with lower magnification, slides were automatically digitized using an Aperio CS2 slide scanner (Leica Biosystems), and image files were generated using the Image Scope Software (Leica Biosystems).
RNA isolation and reverse transcription quantitative polymerase chain reaction RNA was extracted from 25 mg of lung homogenates and tracheal swabs using the innuPREP Virus RNA Kit (Analytik Jena). SARS-CoV-2 RNA copies were quantified using the NEB Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) on the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) as previously described (42).

Serum virus neutralization assay
Microneutralization assay was done with six sera from hamsters that were mock-vaccinated and challenge-infected with the B.1 virus variant, and with five, six, and six sera from hamsters that were sCPD9-vaccinated and challenge-infected with the B. Microneutralization assay was performed by twofold serial dilutions (1:16 to 1:2048) of complement-inactivated (56°C, 30 min) hamster serum plated on subconfluent monolayers of Vero E6 cells in 96-well plates. Cells in each well were infected with 100 PFUs of the respective SARS-CoV-2 variant, incubated for 72 hours at 37°C, fixed with 4% formalin for 24 hours, and stained with crystal violet (0.75% aqueous solution). Serum neutralization was considered effective in wells that showed no any cytopathic effect, and the last effective dilution was counted.

SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at https://science.org/doi/10.1126/ sciadv.abk0172 View/request a protocol for this paper from Bio-protocol.