Cells

Vero clone E6 was purchased from ATCC (ATCC# CRL-1586, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich, Steinheim, Germany) containing 10% fetal bovine serum (FBS; Sigma Aldrich, Steinheim, Germany) and 2 mM l-glutamine (l-Gln, Sigma Aldrich). Cell cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ up to 30 passages after thawing.

Viruses

SARS-CoV-2 (isolate MUC-IMB1)⁵² was a kind gift of G. Dobler (Bundeswehr Institute for Microbiology, Germany) and was used in passage 3 after isolation from a patient for infection and passage 4 for titration of nAb titers²³. SARS-CoV-2 variants B1.617.2 (Delta lineage) or B.1.1.529 (Omicron lineage) were used in passage 5 or passage 3 after isolation from a patient, respectively⁵³.

Syrian golden hamster animal model

All animal experiments were carried out in compliance with the regulations of German animal protection laws and as authorized by the RP Darmstadt and reported according to the ARRIVE guidelines. Six to 12-week-old Syrian golden hamsters (Envigo RMS, Venray, Netherlands) were randomized for sex-matched groups of four animals. Animals were vaccinated intramuscularly (i.m.) in a prime-boost schedule (Day 0 and 21) with 20 μg Comirnaty (BioNTech, Mainz)²⁷, 4 μg Spikevax (Moderna)¹⁶, 1 × 10¹⁰ vp Jcovden (Johnson & Johnson)²¹ or 0.5 × 10⁸ IU Vaxzevria (AstraZeneca)³³. These doses were used previously in the respective animal models, exceeding the equivalent human dose based on body weight. Additionally, four animals were vaccinated subcutaneously (s.c.) with 10 µg recombinant SARS-CoV-2 S protein of the original Wuhan lineage (Cat.-No. 40589-V08B1; Sino Biological Europe, Eschborn, Germany) adjuvanted with 500 µg aluminum hydroxide (Allhydrogel adjuvant 2%, vac-alu-250, InvivoGen, San Diego, CA, USA). For approved human vaccines, clinical lots of Comirnaty, Spikevax, and Jcovden were used before the end of shelf-life. For Vaxzevria, a clinical lot was used that had reached the end of shelf-life 20 d or 41 d before the first or the second immunization, respectively. Hamsters inoculated with 100 µl PBS i.m. served as control animals. All vaccinations were performed under transient narcosis with isoflurane (4%) and a final volume of 100 μL vaccine per animal and application. Blood was drawn on days 0, 21, and 35 under transient narcosis with isoflurane (4%). Vaccinated hamsters were challenged on day 35 by applying 4 × 10³ TCID₅₀ SARS-CoV-2 (isolate MUC-IMB1) of passage 3 in 100 µl volume intranasally under narcosis with ketamin-medetomidine (i.p.: 100 mg/kg body weight ketamine; 0.25 mg/kg body weight medetomidine), antagonized after the manipulations by Atipamezol (s.c.: 0.25 mg/kg body weight). Hamsters were monitored and sacrificed 4 days after infection by overdosing with ketamine-medetomidine (i.p.: 300 mg/kg body weight Ketamin; 0.75 mg/kg body weight medetomidine), and lung tissue was prepared for histology (left lobe), analysis of total RNA (right middle lobe) and titration of live virus (right apical lobe). Table 1

Bronchoalveolar lavage (BAL)

Bronchoalveolar lavage (BAL) was performed as previously described¹⁵ using 2.5 mL PBS applied and excised via the trachea. Cells were harvested by centrifugation (1200 rpm, 4 °C, 5 min) and resuspended in 350 μL TRIzol Reagent (Ambion, Thermo Fisher Scientific).

Total IgG quantification

Quantification of binding antibodies via ELISA was performed as previously described¹⁵ using 0.25 μg recombinant unmodified SARS-CoV-2 S protein of the original Wuhan lineage (Sino biologicals) in carbonate buffer (Na₂CO₃ 30 mM; NaHCO₃ 70 mM; pH 9.6) to coat Nunc Maxisorp® 96-well ELISA plates (eBioscience) and HRP-conjugated goat anti-hamster IgG (1:1000 in PBS containing 1% BSA and 0.1% Tween20; Sera care KPL, Cat. 5220-0371) for detection of antigen binding antibodies in the analyzed hamster sera using TMB substrate (eBioscience). After stopping the reaction with 1 N H₂SO₄, the absorbance at 450 nm (specific signal) and 630 nm (reference wavelength) was measured.

Virus neutralization test (VNT)

A virus neutralization test (VNT) was performed as described previously^15,23. Shortly, 100 TCID₅₀ SARS-CoV-2 (MUC-IMB1, B1.617.2 or B.1.1.529) were mixed with 2-fold serially diluted serum samples and incubated at 37 °C for 1 h. Subsequently, the virus-serum mixture was added to 1 × 10⁴ Vero E6 cells seeded in 96-well plates 3 h before. Cells were incubated for 4 days at 37 °C in a humidified atmosphere containing 5% CO₂. The virus-neutralizing titer was determined by the reciprocal of the highest serum dilution that prevent CPE formation (MUC-IMB1, B1.617.2) or infection determined by immunofluorescence staining (B.1.1.529).

Immunofluorescence staining assay (IFA)

For the determination of cell cultures infected by SARS-CoV-2 Omicron variant B.1.1.529, the medium of the cells was removed, and the cells were washed with PBS. The cells were then fixed in 4% PFA overnight prior to being permeabilized with 0.5% Triton X-100 for 5 min at room temperature (RT). After washing with PBS, the cells were blocked with 10% goat serum (Abcam, Cat.-No. ab7481) in PBS for 30 min at RT. The cells were then washed and incubated with an anti-SARS-CoV-2 nucleocapsid protein antibody (1:2000; antibodies-online, Cat-No. ABIN6952544) in 2% goat serum in PBS for 1.5 h at RT. After washing with PBS, the cells were incubated with a Cy5-anti-rabbit IgG antibody (1:1000; Invitrogen, Cat-No. A10523) in PBS containing 2% goat serum for 1 h at RT. After a final wash with PBS, fluorescence was assessed under a microscope (ECHO Revolve, ECHO, San Diego, CA, USA).

Determination of lung virus titers in infected animals

The right apical lobe of the lung was snap-frozen in liquid nitrogen and homogenized in 1 mL ice-cold DMEM containing 2 mM l-Gln and 1% Penicillin/Streptomycin as previously described¹⁵ using Precellys24 tissue homogenizer (bertin TECHNOLOGIES, Montigny-le-Bretonneux, France) and Lysing Matrix M tubes (MP Bioscience, Hilton, UK). The tenfold serially diluted supernatant was used to inoculate Vero E6 cells for 7 d in a humidified atmosphere at 37 °C. SARS-CoV-2 organ titer was calculated by the TCID₅₀ method of Kaerber and Spearman according to virus-induced CPE and normalized to 1 g of homogenized tissue.

RNA preparation

The right middle lobe of the lung was homogenized in 1 mL TRIzol Reagent (Ambion, Thermo Fisher Scientific) as previously described¹⁵ using Precellys24 tissue homogenizer (bertin TECHNOLOGIES) and in Lysing Matrix M tubes (MP Bioscience). Cell debris was removed by subsequent centrifugation (6800 rpm; 4 °C; 13 min), and the clear supernatant was used for RNA purification with a Direct-zol RNA MiniPrep kit (Zymo Research, Freiburg (Breisgau), Germany) according to the manufacturer’s instructions.

Determination of virus genome copy numbers SARS-CoV-2 Egene and hamster RPL18 by qPCR

Quantification of viral RNA in the samples was performed by quantitative reverse transcription-PCR (qRT-PCR) using a Superscript III one-step RT-PCR system with Platinum Tag Polymerase (Invitrogen, Darmstadt, Germany). Primer and probe sequences for SARS-CoV-2 Egene⁵⁴, IL-4⁵⁵, IL-13⁵⁵, IFN-γ⁵⁵, and the RPL18 housekeeping gene⁵⁶ were used as described previously (Table 1).

Each sample was analyzed in triplicates in a 96-well plate format using the CFX96 qPCR cycler (Bio-Rad Laboratories, Hercules, CA) and 5 µl RNA in a total reaction volume of 25 µl. Quantification of the copy numbers of the SARS-CoV-2 E gene was carried out using an internal hamster reference (linear range, 4.5 × 10⁶ to 4.5 × 10² copies²³). This reference was validated for copy numbers of RPL18 by using a PCR product DNA reference prepared as described previously⁵⁷, and was used for quantification in subsequent runs (linear range, 1.8 × 10⁵ to 1.82 × 10²). The following cycling conditions were used: reverse transcription for 600 s at 55 °C, denaturation for 180 s at 94 °C, followed by 45 cycles of 15 sec at 94 °C and 30 s at 58 °C. Quantified E gene copy numbers were normalized to copy numbers of the RPL18 housekeeping gene.

Histopathological examination

The left lung lobe was fixed in 4% formalin for 7 days. The tissue was subsequently paraffin-embedded, and sections of 4 μm were prepared.

Hematoxylin-eosin staining

Hematoxylin-eosin staining was carried out in accordance with standard protocols⁵⁸. H&E stained slices were subjected to histopathologic analyses on blinded samples.

Sirius Red staining

For Sirius Red staining of lung tissue sections we used a modified protocol published by Llewellyn that was used previously^15,59. First, sections were soaked in Papanicolaous solution 1b Hematoxylin S (Sigma Aldrich) for 2 min and then rinsed with water, ethanol, 3% HCl in ethanol, and 70% ethanol. Sections were then stained in alkaline Sirius red (0.5 g Direktrot 80, Sigma in 50% ethanol containing 0.1‰ NaOH) for 90 min before being rinsed with water. Subsequently, sections were dehydrated with increasing concentrations of ethanol and xylene. Finally, the sections were covered with Entellan (Merck KGaA, Darmstadt, Germany).

In situ hybridization

To detect viral RNA in the lung tissue sections, fixed paraffin-embedded tissue sections were mounted on glass slides and analyzed by in situ hybridization as described previously^60,61. The RNAscope® 2.5 HD Assay—RED Kit (Bio-Techne, cat. no. 322360) was used according to the manufacturer’s instructions. To gain access to the target RNA, slides were incubated at 60 °C, deparaffinized with xylene and 100% ethanol, and pretreated with RNAscope® Pretreatment Reagents (cat. no. 322330 and 322000). Subsequently, the RNA-specific probe, targeting the S protein of the SARS-CoV-2 virus (cat. no. 848561), was hybridized to the RNA. After the amplification steps, Fast Red substrate was added to the samples for signal detection. Slides were counterstained with Gill’s Hematoxylin I and 0.02% ammonia water. An RNAscope® Negative Control Probe (cat. no. 310043) was used in parallel to control background staining.