Hamster Model of Severe COVID-19 Demonstrates Differential Efficacy of first Licensed Western Vaccines in the Absence of Immunopathogenesis

doi:10.21203/rs.3.rs-4151277/v1

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Hamster Model of Severe COVID-19 Demonstrates Differential Efficacy of first Licensed Western Vaccines in the Absence of Immunopathogenesis

https://doi.org/10.21203/rs.3.rs-4151277/v1

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To combat the COVID-19 pandemic, vaccines have been developed, tested and approved in less than one year. Four vaccines using two technology platforms were authorized early in Europe and the US. Comirnaty and Spikevax are mRNA-based, whereas Jcovden and Vaxzevria utilize adenoviral vectors (AdV). We already described a hamster model of severe COVID-19, in which vaccine-associated immunopathogenesis can be induced by Alum-adjuvanted Spike protein. Such animals were vaccinated here with the early authorized vaccines, challenged, and examined for immunopathogenesis in comparison to Alum+S-vaccinated or naïve hamster after challenge. All vaccinated hamsters produced antibodies binding (bAb) to SARS-CoV-2 Spike, while neutralizing antibodies (nAb) were induced only by the authorized vaccines. Among those, uniform induction of nAbs by mRNA vaccines needed a second dose. Using AdV-based vaccines, nAbs were mostly abundant after just one vaccination, but boostability was low. Upon challenge, Alum+S immunized animals were not protected and developed VAERD. In contrast, all authorized vaccines protected from severe disease, but transient initial weight loss was observed. In accordance with lower nAb titers, a tendency for higher weight loss became evident with Vaxzevria. Histopathology revealed less tissue damage after immunization with the authorized vaccines, with two animals revealing unaffected, healthy lungs. No live virus was detectable in lung tissue in those vaccine groups, except for one animal with Vaxzevria. Our data reveal absence of induction of VAERD by the authorized vaccines in a susceptible hamster model, while the induced immune responses and observed degree of protection seem to match the clinical vaccine efficacy.

Health sciences/Diseases/Infectious diseases/Viral infection

Health sciences/Medical research/Experimental models of disease

Biological sciences/Microbiology/Vaccines

Biological sciences/Immunology/Vaccines

Health sciences/Pathogenesis/Immunopathogenesis/Adaptive immunity

SARS-CoV-2

COVID vaccines

COVID-19 hamster model

immunopathogenesis

vaccine comparison

To combat the COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a number of different vaccines have been developed, tested, and licensed in a remarkably short time. To date, more than 13 billion doses (March 12 2024 ^⁠¹) of those authorized vaccines have been administered. These vaccines were developed on the basis of traditional approaches such as inactivated virus or adjuvanted, recombinant proteins, but especially in the Western World by novel technologies, i.e., mRNA vaccines or adenoviral vectors. Remarkably, all of the latter target the Spike protein S of SARS-CoV-2, which is the target of neutralizing antibody responses due to its role in receptor binding and cell entry during the pathogen life cycle.

One potential safety risk in vaccine development is enhancement of the disease in vaccinated individuals later infected with the respective pathogen due to induction of immune responses that facilitate development of immunopathologies. Enhancement of disease by vaccine-induced syndromes such as vaccine-associated enhanced respiratory disease (VAERD) or antibody-dependent enhancement (ADE) has been described for a number of different viral diseases such as infections with dengue virus ^⁠2, respiratory syncytial virus ^⁠3–6, or feline infectious peritonitis virus ^⁠7–9. Accordingly, animal models revealed the potential risk of VAERD for infections with the first two highly pathogenic human beta-CoVs, SARS-CoV or Middle East respiratory syndrome (MERS)-CoV. After superinfection of receptor-transgenic mice previously immunized with whole-inactivated virus, the animals developed severe lung pathology with massive infiltration of eosinophils ^10–12. Such enhanced immunopathology has been linked to induction of low-affinity and non-neutralizing antibody responses ^⁠6,7 or vaccine-induced T_H2-biased T cell responses ^⁠5.

Although such enhancement processes have not been described for SARS-CoV-2 in human vaccinees, typical features of VAERD have been observed in T_H2-biased mice vaccinated with alum-adjuvanted whole inactivated virus or S-protein upon subsequent infection with mouse-adapted SARS-CoV-2 ^13,14. Similarly, we detected and proposed a detailed mechanism for the induction of VAERD in outbred hamsters vaccinated with Alum-adjuvated S protein upon subsequent infection with a low-passage human patient isolate of SARS-CoV-2. Our model suggests that induction of a T_H2-specific S-specific T-cell response and the induction of low level of non-neutralizing antibodies leading to Fc-receptor mediated uptake of the virus into lung macrophages are the main causes of the development of VAERD ¹⁵. Absence of such reactions may be attributed at least in part by the design of most of the authorized vaccines the construction of which considered this potential risk by avoiding induction of T_H2-biased immunity ^16–22.

Here, we have utilized our hamster model of severe COVID-19 ²³ to analyze side-by-side the protective effects of the four vaccines, which received early approval in the European Union and the United States: Comirnaty (BioNTech) ¹⁹ and Spikevax (Moderna) ^16,17 are based on mRNA vaccine technology while Jcovden (Johnson & Johnson) ^20–22 and Vaxzevria (AstraZeneca) ¹⁸ utilize the adenovirus vector (AdV) platform technology. To our knowledge, our study presented here is the first comparing all four commercial vaccines derived from two very different vaccine platform technologies in a relevant animal model for COVID-19, simultaneously, allowing assessment of vaccine efficacy and potential adverse events side-by-side.

All four vaccines protected the hamsters after twofold vaccination from severe disease without showing evidence of eosinophil infiltration as a hallmark of immunopathogenesis. Only one out of 16 hamsters had low titer infectious virus in the lungs, while viral RNA copy numbers were around the limit of detection in lungs and BAL fluid. This protection correlated with induction of considerable nAb responses by all tested authorized vaccines. As a remarkable difference between vaccine concepts, both AdV-derived vaccines induced nAb titers already after one vaccination, while the second vaccination with the mRNA vaccines fully compensated this initial difference.

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) ²⁴ was a kind gift of G. Dobler (Bundeswehr Institute for Microbiology, Germany) and was used in passage 3 after isolation from a patient ²³.

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-weeks old Syrian golden hamsters (Envigo RMS, Venray, Netherlands) were randomized for sex-matched groups of four animals. Animals were vaccinated intramuscular (i.m.) in a prime-boost schedule (Day 0 and 21) with 20 µg Comirnaty (BioNTech, Mainz) ²⁵, 4 µg Spikevax (Moderna) ¹⁶, 1 x 10¹⁰ vp Jcovden (Johnson & Johnson) ²¹ or 0.5 x 10⁸ IU Vaxzevria (AstraZeneca) ²⁶. Additionally, four animals were vaccinated subcutaneously (s.c.) with 10 µg recombinant SARS-CoV-2 S protein (Sino Biological Europe, Eschborn, Germany) adjuvanted with 500 µg aluminum hydroxide (Allhydrogel adjuvant 2%, vac-alu-250, InvivoGen, San Diego, CA, USA). Hamsters inoculated with 100 µl PBS i.m. served as control animals. Blood was drawn on days 0, 21, and 35. Vaccinated hamsters were challenged on day 35 by applying 4 x 10³ TCID₅₀ SARS-CoV-2 (isolate MUC-IMB1) in passage 3 in 100 µl volume intranasally. Hamsters were monitored and sacrificed 4 days after infection and lung tissue was prepared for histology (left lobe), analysis of total RNA (right middle lobe) and titration of live virus (right apical lobe).

Bronchoalveolar lavage (BAL)

Bronchoalveolar lavage (BAL) was performed as previously described ¹⁵ using 2.5 mL PBS applied and excised via the trachea. Cells were harvested by centrifugation (1,200 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 described ¹⁵ using 0.25 µg recombinant SARS-CoV-2 S protein (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 (1:1,000 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 1N H₂SO₄ the absorbance at 450 nm (specific signal) and 630 nm (reference wavelength) was measured.

Virus neutralization test (VNT)

Virus neutralization test (VNT) was performed as described previously ^15,23. Shortly, 100 TCID₅₀ SARS-CoV-2 (MUC-IMB1) 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 x 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.

Determination of lung virus titers in infected animals

The right apical lope 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 described ¹⁵ using Precellys24 tissue homogenizer (bertin TECHNOLOGIES, Montigny-le-Bretonneux, France). The 10-fold 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 1g 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 described ¹⁵ using Precellys24 tissue homogenizer (bertin TECHNOLOGIES) and in Lysing Matrix M tubes (MP Bioscience, Hilton, UK). Cell debris was removed by subsequent centrifugation (6800 rpm; 4°C; 13 min) and clear supernatant was used for RNA purification with 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 Superscript III one step RT-PCR system with Platinum Tag Polymerase (Invitrogen, Darmstadt, Germany). Primer and probe sequences for SARS-CoV-2 Egene ²⁷ and the RPL18 housekeeping gene ²⁸ were used as described previously.

E_Sarbeco_F	Corman et al. 2020	ACAggTACgTTAATAgTTAATAgCgT
E_Sarbeco_R	Corman et al. 2020	ATATTgCAgCAgTACgCACACA
E_Sarbeco probe	Corman et al. 2020	5‘-(6FAM)-ACACTAgCCATCCTTACTgCgCTTCg(BBQ)-3‘
RPL18 F	Zivcec et al. 2011	5‘-GTTTATgAgTCgCACTAACCG-3‘
RPL18 R	Zivcec et al. 2011	5‘-TGTTCTCTCggCCAggAA-3‘
RPL18 probe	Zivcec et al. 2011	5‘-(Cy5)-TCTgTCCCTgTCCCggATgATC(BBQ)-3‘

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 SARS-CoV-2 E gene was carried out using an internal hamster reference (linear range, 4.5 x 10⁶ to 4.5 x 10² copies, ²³). This reference was validated for copy numbers of RPL18 by using a PCR product DNA reference prepared as described previously ²⁹, and was used for quantification in subsequent runs (linear range, 1.8 x 10⁵ to 1.82 x 10²). Following cycling conditions were used: reverse transcription for 600 sec at 55°C, denaturation for 180 sec at 94°C, followed by 45 cycles of 15 sec at 94°C and 30 sec at 58°C. Quantified E gene copy numbers were normalized to copy numbers of 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.

Hematoxilin-Eosin staining

Hematoxylin-eosin staining was carried out in accordance with standard ³⁰. 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 ^15,31. 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 (0.5g 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 hybridisation

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 ^32,33. 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 % etanol 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 % amonia water. A RNAscope® Negative Control Probe (cat. no. 310043) was used in parallel to control background staining.

Protection by authorized COVID-19 vaccines in the immunopathogenesis-prone hamster infection model.

Groups of six to 12 week-old Syrian golden hamsters (n = 4) were immunized side-by-side with adapted doses of authorized COVID-19 vaccines Comirnaty, Jcovden, SpikeVax, or Vaxzevria on days 0 and 21 via the intramuscular (i.m.) route (Fig. 1A). To control for inducibility of immunopathogenesis, recombinant SARS-CoV-2 Spike protein adjuvanted with aluminum hydroxide gel (Alum + S) was injected subcutaneously in a separate cohort by the same schedule, as described before ¹⁵. Medium-inoculated hamsters served as naïve controls (mock). Two weeks after the second vaccination, all hamsters were challenged intranasally (i.n.) with a low-passage SARS-CoV-2 patient isolate. Animals were monitored for 4 days, sacrificed, and lungs were prepared for histopathology, analysis of viral RNA copy numbers, and titration of live virus.

As previously described, hamsters immunized with Alum + S were not protected and lost weight upon infection with the same kinetics as naïve control animals (Fig. 1B, C). Moreover, the mean weight of the lungs (2.088 g) was increased as compared to naïve animals (1.694 g), indicating immune cell infiltration and exaggerated inflammatory processes, as also suggested by gross macroscopic analysis of the lungs (Fig. 1E, G). In contrast, most animals in all vaccine cohorts recovered after initial weight loss after day 2 post infection. Lung weights were significantly reduced compared to the Alum + S cohort (Fig. 1E, F) and nearly reached significance for the Comirnaty group also in comparison to naïve infected animals. Comirnaty, Jcovden, or Spikevax-immunized animals had comparable mean lung weights between 1.319 g to 1.417 g, while the Vaxzevria cohort revealed a slightly higher mean lung weight of 1.589 g, although this difference did not reach statistical significance. These observations came along with reduced pathological alterations of lungs on the macroscopic (Fig. 1G, Suppl. Fig. S1) as well as microscopic level (Suppl. Figure 3A).

In these animals, nearly no live virus was detected in lung tissues, except for one animal from the Vaxzevria group with a low titer of 1.6 x 10³ TCID₅₀/g (Fig. 2A). However, also hamsters immunized with Alum + S revealed lower virus loads in the lung compared to naïve, infected mock animals (Fig. 2A), again indicating the immunopathological mechanism of disease in these animals. Analysis of relative abundance of viral RNA in lung tissue (Fig. 2B) and bronchoalveolar lavages (Fig. 2C) were in accordance with live titers: In the cohorts, which had been vaccinated with Comirnaty or Jcovden, at least half of the analyzed samples were below the limit of detection. Three out of four animals immunized with Spikevax had low, but detectable viral RNA load (median 0.227 E / RPL18 gene copies). Only in the Vaxzevria cohort, all samples revealed a low, but detectable RNA load about 500-fold lower than naïve control animals (median 0.134 vs. 458.17 E / RPL18 gene copies). In contrast, Alum + S immunized animals also had a lower RNA load (median 45.61 E / RPL18 gene copies) detectable in all animals, but reduction was thereby just about tenfold.

In situ hybridization (ISH) indicates active SARS-CoV-2 infection in tissues with pathological alterations.

As already indicated by the detection of viral RNA by qRT-PCR, active SARS-CoV-2 infection in lungs of the infected animals was demonstrated by ISH of SARS-CoV-2 RNA genomes in lung tissue (Fig. 3A, third row and 3D). As previously reported, Alum + S vaccinated, infected animals showed reduced staining for SARS-CoV-2 RNA genomes (mean area score 0.375) as compared to infected mock-immunized hamsters (mean area score 1.125). All four vaccine cohorts showed reduced staining for SARS-CoV-2 RNA genomes compared to naïve, infected animals to a different extent (mean area scores: Comirnaty 0.25; Jcovden 0.25; Spikevax 0.5; Vaxzevria 0.375). Interestingly, only in the Spikevax group, all animals had detectable area score for SARS-CoV-2 RNA genomes.

Absence of immunopathogenesis upon immunization with authorized vaccines.

In accordance with our previous report ¹⁵, histological analysis of hematoxylin and eosin (H&E)-stained lung tissue slices revealed the replication of the expected pathology for the control animals. In naïve, infected hamsters, moderate inflammation of the epithelia and endothelia in bronchii and vasculature was observed (Table 1). Moderate infiltration of macrophages and lymphocytes resulted in a fraction of 1%-50% of dense areas (Table 1). Among the immune cell infiltrates, no or few eosinophils or granulocytes were observed, with the exception of a single animal showing significant infiltration of granulocytes. Individual foci of moderate karyorrhexis became also evident in these animals. In contrast, Alum + S-vaccinated, infected hamsters revealed an intensified pathology (Fig. 3A, B). The areas of dense infiltrates varied between 20% and 50% of the tissue slices (Suppl. Fig. S2, Table 1). Large areas of inflammation of bronchii and vasculature included massive infiltration of eosinophils (Fig. 3C).

Table 1

**Histopathological analysis of lung tissue of vaccinated Syrian hamsters upon challenge with SARS-CoV-2.** Hamster were vaccinates with medium (MOCK), Alum-adjuvanted Spike protein (Alum + S), Comirnaty, Spikevax, Jcovden or Vaxzevria. The left lope of vaccinated hamster lungs were dissected 4 dpi with low passage SARS-CoV-2.
Animal No.	Vaccine Group	% Dense Area	Bronchia	Vessels	Dense Area
124	MOCK	about 20% dense area, bronchial associated	Bronchial epithelium overall only slightly affected, proliferation; little inflammatory infiltration; scattered desquamated cells in the lumen; isolated karyorrhexis; NO bronchial epithelium in the surrounding area	Vascular walls with sporadic inflammatory infiltration; proliferation of the endothelium; sporadic karyorrhexis; hemorrhages	few dense areas, with distinct infiltration by macrophages, lymphocytes, and granulocytes
128	MOCK	about 40% slightly dense areas, no bronchial association	Bronchial epithelium proliferated, rejected cells in the lumen, mild inflammatory infiltration of the epithelium.	Endothelium of the vessels rounded; otherwise without findings	Areas only moderately condensed, structure of lung still recognizable. Infiltration of macrophages and lymphocytes, no granulocytes, no eosinphils
140	MOCK	about 50% dense area, not bronchial associated	Proliferation of epithelium; epithelium partially destroyed; sporadic granulocytes in lumen	sporadic granulocytes in the vascular wall, hemorrhages	Areas only moderately compacted, structure of lung still recognizable. Infiltration with macrophages and lymphocytess, no granulocytes, no eosinophils
143	MOCK	max. 1% dense area	Bronchi only slightly/not affected	Endothelium of the vessels rounded; otherwise without special findings.	Structure of lungs clearly preserved, only minimally dense areas, no macrophages, lymphocytes, many granulocytes, no eosinophils.
121	Alum + S	about 20% dense areas, bronchial or vascularly associated	Bronchial epithelium with mild inflammatory infiltration; eosinophilic granulocytes in the lumen; few bronchial epithelium in the surrounding tissue	Vascular walls sporadically infiltrated with inflammation; (including eosinphils), sporadic bleeding	few dense areas, with distinct infiltration by macrophages, pneumocytes, lymphocytes and massive infiltration by eosinophilic granulocytes
125	Alum + S	40% dense area, only partially vascularly associated	no clear bronchi recognizable; massive bronchial epithelium in the surrounding tissue.	Vascular walls clearly affected; infiltration with eosinophils; karyorrhexis; sporadic hemorrhages	One large condensed area; massive infiltration of bronchial epithelium, areas with distinct eosinophils, lymphocytes and marcophages (pneumocytes); areas with distinct karyorrhexis
129	Alum + S	50% dense area, no longer bronchial associated	Bronchial epithelium clearly involved, partially destroyed. Inflammatory infiltration. Granulocytes and eosinophils in the lumen.	Vascular walls clearly affected; infiltration with eosinophils; sporadic distinct hemorrhages	massively condensed areas, infiltration with bronchial epithelium, pneumocytes, macrophages, lymphocytes, granulocytess and many eosinophils
133	Alum + S	about 50% dense areas, not bronchial associated	proliferation of the epithelium, granulocytes in the lumen	Inflammatory cells in the vascular wall	moderate dense areas; infiltration with pneumocytes, macrophages, lymphocytes, granulocytes and some eosinophils
122	Comirnaty	Less than 10%, bronchial or vascularly associated	Bronchial epithelium not affected	Vascular walls not affected; perivascular low lymphatic infiltration	minimally dense areas with distinct infiltration by macrophages and lymphocytes; no granulocytes. Very low karyorrhexis.
126	Comirnaty	max. 5% dense area, bronchial associated	Bronchial epithelium inflammatory infiltrated, some inflammatory cells in the lumen, bronchial epithelium in the surrounding area	Vessels not affected	small dense areas with infiltrated bronchial epithelium, macrophages and lymphocytes, few granuloctes
130	Comirnaty	no dense areas	Bronchi not affected	Vessels not affected	none
134	Comirnaty	about 25% dense area, not bronchial associated	Slight proliferation of the epithelium	Rounding of endothelium, mild edema around vessels, hemorrhage	Distinct condensed areas, infiltration with pneumocytes, macrophages, lymphocytes, few granulocytes and a few eosinophils.
123	Spikevax	about 30% dense area (many small foci), clearly bornchial and vascular associated.	Bronchial epithelium slightly infiltrated with inflammation; lymphocytes and sporadic eosinophilic granulocytes in the lumen, clearly bronchial epithelium in the surrounding area	Vessel walls sporadically infiltrated with inflammation, (including eosinphils), significant bleedings, clear perivascular infiltrates	clearly dense areas, these clearly infiltrated with macrophages, pneumocytes, lymphocytes, but hardly any granulocytes
127	Spikevax	max. 10% dense area, strictly bronchial associated	Bronchi not affected	Vessels not affected	small dense areas with infiltrated bronchial epithelium, macrophages and lymphocytes, some eosinophils, sporadic karyorrhexis
137	Spikevax	max. 1% dense area; only one very small condensed area on a vessel	Bronchi not affected	Vessels not affected	Macrophages, lymphocytes and sporadic eosinophils
141	Spikevax	max. 1% dense area	Bronchi only slightly/not affected	Endothelium of the vessels rounded; otherwise without special findings	Structure of lungs clearly preserved, only minimally dense areas, no macrophages, many lymphocytes, single eosinophils
131	Jcovden	max. 5% dense area, bronchial associated	Only 1 bronchus affected, with few inflammatory cells in epithelium, exfoliated cells in the lumen	Endothelium of the vessels rounded; otherwise without findings	One distinct dense area; massive infiltration of bronchial epithelium, areas with distinct infiltration of lymphocytes and macrophages (pneumocytes), no granulocytes, no eosinophils, no distinct karyorrhexis
135	Jcovden	about 40% dense area, not bronchial associated	Only slightly affected, slight focal proliferation of the epithelium	some inflammatory cells in the vascular wall, edema around vessels	Distinct condensed areas, infiltration with a lot of pneumocytes, macrophages, fewer lymphocytes, few granulocytes and some eosinophils
139	Jcovden	no dense areas	Bronchi not affected	Vessels not affected	none
144	Jcovden	1% dense area, vascular	Bronchial epithelium in the surrounding area partially destroyed (focal)	Vessels not visibly affected	Structure of lung preserved; only minimal dense areas; many macrophages in infiltration of bronchial epithelium, lymphocytes, no granulocytes, some eosinophils
132	Vaxzevria	about 1% dense area, bronchial associated	only 1 bronchus clearly affected, proliferating epithelium, exfoliated cells in the lumen	rounded endothelium, inflammatory cells (also sporadic eosinophils) in the vicinity of the vessel	3 small dense areas with lymphocytes, macrophages, granulocytes and eosinophils
136	Vaxzevria	about 20% dense area, bronchial associated	Only slightly affected, slight focal proliferation of the epithelium	Endothelium of the vessels rounded; otherwise without special findings	Distinct dense areas, infiltration with pneumocytes, macrophages, lymphocytes, few granulocytes and und focal distinct eosinophils
138	Vaxzevria	about 25% dense area, not bronchial associated	Only slightly affected, slight focal proliferation of the epithelium, but infiltration into the dense areas	Vessels slightly affected	Distinct dense areas; infiltration with pneumocytes, macrophages, lymphocytes, few granulocytes and a few eosinophils
142	Vaxzevria	about 40% dense area, not bronchial associated	in the dense areas no healthy bronchi bronchi visible; instead, massive infiltration of the epithelium into the surrounding area, blood in the bronchi at the edge.	Vessels not visibly affected, but massive bleeding in the tissue	Dense areas of infiltration with many pneumocytes, macrophages; infiltration of bronchial epithelium, fewer lymphocytes, no granulocytes and no eosinophils

On the other hand, lung tissue derived from hamsters vaccinated with mRNA-based vaccines Comirnaty or Spikevax revealed reduced inflammation, with only 0–10% of the tissue becoming dense in most animals (Table 1). Only a single hamster, each, immunized either with Comirnaty or Spikevax exhibited 25% and 30% dense areas, respectively (Table 1). Most animals revealed low to no eosinophilic infiltrates in the lungs (Fig. 3C). Thus, the pathological score was considerably reduced for both groups, with one animal in the Comirnaty group scoring as completely “healthy” (Fig. 3B). Hamsters vaccinated with AdV-based vector vaccines Jcovden or Vaxzevria revealed 1–40% dense areas. Interestingly, three out of four animals in the Jcovden group had between 0% and 5% dense areas, again one animal scoring “healthy” (Fig. 3B). However, another Jcovden hamster had up to 40% dense areas. On the other hand, three out of four animals receiving Vaxzevria had between 20–40% dense areas. Here, a single animal in this group had only about 1% compacted tissue. Again, no to low numbers of eosinophils were observed in the lung tissue (Fig. 3A).

Sirius Red staining for eosinophils confirmed significant infiltration of eosinophils in infected hamsters inoculated with Alum + S, as already observed in H&E-stained lung sections. In these animals, between 190 and 340 eosinophils/mm² (mean 275.1 eosinophils/mm²) were found. In contrast, the mean number of eosinophils was reduced 2.6- to 7.6-fold (mean: 35.9–104.3 eosinophils/mm²) in hamsters vaccinated with the authorized COVID-19 vaccines. Only one animal from each, the Comirnaty (291.6 eosinophils/mm²), Spikevax, or Vaxzevria group (both 148.8 eosinophils/mm²) had a higher eosinophil density (Fig. 3C).

Thus, these data confirm protection of immunized animals in absence of any signs of VAERD in hamsters vaccinated with the authorized COVID-19 vaccines, in a severe COVID-19 hamster infection model capable of revealing pathological alterations indicative of vaccine-induced immunopathogenesis.

Protection correlates with induction of nAbs, immunopathogenesis with lower titers of bAbs in absence of nAbs.

14 days after the second immunization, sera were analyzed for binding antibodies (bAbs) targeting S by ELISA in comparison to pre-bleed and post-prime sera. Binding antibodies were induced after the primary vaccination for all vaccine groups and boosted by the second immunization for the mRNA vaccines but only moderately for the AdV-based vaccines. In contrast no SARS-CoV-2 Spike bAbs were detected in pre-bleed sera or sera derived from MOCK-immunized animals. All authorized vaccines induce comparable amounts of SARS-CoV-2 Spike bAbs. In contrast, the experimental Alum + S vaccine induced lower amounts of bAbs in comparison to the authorized vaccines. (Fig. 4A).

We also determined neutralizing antibody responses to SARS-CoV-2 by virus neutralization test (VNT) (Fig. 4B). Neutralizing antibodies (nAb) were detected in sera of all hamsters that were immunized with authorized vaccines after boost immunization, whereas hamsters that received Alum + S showed only S-binding antibodies without neutralizing activity, as previously reported ^15,23. Uniform induction of nAbs by mRNA vaccines was observed after the second dose; only serum from a single animal in the Spikevax group lacked detectable nAb titers after the boost immunization. On the other hand, a single hamster in the Comirnaty group showed low nAb titers already after a single dose of the vaccine. Vaccination with the AdV-based vaccines induced nAb in most animals (4/4 for Jcovden and 3/4 for Vaxzevria) already after prime immunization, but titers increased only marginally after the second shot. Nevertheless, also the 4th Vaxzevria animal became positive for nAbs after the boost immunization. Overall, nAb titers appeared to be slightly lower in animals vaccinated with Vaxzevria compared those immunized with the other authorized vaccines (Fig. 4B).

Taken together, our data show that VAERD does not occur in SARS-CoV-2-infected hamsters immunized with all licensed vaccines tested, due to the absence of eosinophils in tissue sections, a marker for VEARD. All vaccines tested significantly reduced viral load in the lung and no significant differences were observed between the different vaccine cohorts. However, animals vaccinated with Vaxzevria appeared to have a slightly higher lung weight and viral load compared to the other three vaccine cohorts, but still reduced when compared to the naïve, infected hamsters.

During the COVID-19 pandemic, billions of vaccine doses have been administered and to date VAERD has not been described in vaccinees. However, the potential risk of the induction of VAERD for SARS-CoV-2 and related Corona viruses has been described in different animal models and induction of VAERD has been linked to a T_H2-biased immune response ¹⁰^–15^,34 . Although transferability of these findings to human beings remains unclear, all vaccines authorized in the European Union and the United States were constructed to elicit a predominantly T_H1-biased immune response to exclude the risk of potential induction of VAERD ¹⁶^–21. These vaccines have previously shown effectiveness in mice ¹⁶^,25^,35^,36, Non-human primates ¹⁷^,21^,25^,37^–39, some in Syrian hamsters ²¹^,26^,40^–42 and in clinical trials ¹⁸^–20^,22^,43^–45 . Syrian hamsters infected with SARS-CoV-2 consistently reveal the characteristics of severe COVID-19 and are considered as gold standard for reflecting pathogenesis ⁴⁶. Furthermore, Syrian hamsters show under specific conditions VAERD upon vaccination with T_H2-biased vaccines ¹⁵ in the absence of genetic alterations required for either mice ⁴⁷ or virus ⁴⁸) as necessary in the mouse model, but confirming pathologies observed in those models.

Hence, we tested all four early approved Western vaccines side by side in this animal model and confirmed the absence of VAERD in SARS-CoV-2 infected hamsters vaccinated with these vaccines, while respective control hamsters (Alum+S) developed VAERD. Therefore, our data indicate that absence of reports of VAERD in previously vaccinated Western patients after COVID-19 superinfection are indeed due to the appropriate design of these vaccines and not due to the close similarity of VAERD pathologies and symptoms during severe courses of COVID-19 ⁴⁹ . However, our study is limited to mRNA and AdV-derived vaccines available at the time of initiation of our study. Performance of other vaccine concepts, e.g. Alum-adjuvanted, inactivated COVID-19 vaccines approved for emergency use by the WHO ⁵⁰, needs to be analyzed in future additional studies.

Besides demonstrating safety of all tested vaccine concepts, our data allow comparison of the efficacy of the different vaccines in the same setting. Although all commercially available vaccines tested in our study were protective, as they had demonstrated before in individual pre-clinical and clinical studies, minor differences between the vaccine cohorts were observed during our experiment. Hamsters which received the AdV-based vaccines showed only low increase in binding antibodies by the second immunization while neutralizing antibody titers that were induced already after one shot were boosted. This effect has been previously observed for Jcovden ²¹, also in the hamster model. In contrast, a two dose regime of Jcovden boosted binding and neutralizing antibody titers in non-human primates ³⁹. Those differences were attributed to species-specific effects of the responses against the AdV vaccine backbone after the first immunization affecting the immunogenicity of the boost ²¹. In contrast, all hamster vaccinated with an mRNA vaccine showed strong boosting of binding and neutralizing antibody titers by the second vaccine dose in our study which confirms previous data ¹⁹. Boosting of binding antibody titers by repeated vaccination with mRNA vaccines has already been described in hamsters vaccinated with 5 mg Spikevax twice ⁴². Interestingly, only the serum of a single animal immunized with Comirnaty showed a low neutralizing capacity against SARS-CoV-2 after one shot, in our study, while 7/8 animals among the mRNA vaccine groups developed neutralizing antibodies only after two-fold immunization. This is somewhat consistent with previous data providing evidence that not all (3/15) hamsters immunized with Spikevax developed neutralizing antibodies after prime immunization ⁴². In the end, final nAb titers were comparable to those in animals immunized with AdV-based vaccines, showing no clear advantage for any vaccine technology in a prime-boost regimen concerning humoral responses, while the AdV technology seems advantageous for a one-shot regimen.

Anyway, all authorized vaccines were protective in our hamster model without evidence for immunopathologies, in consistence with clinical findings and protection of human vaccinees. No live virus was recovered from the lungs of any AdV- or mRNA-vaccinated hamster. Previously, live virus was detected in the lungs of 3 out of 12 Jcovden-vaccinated animals 4 days after infection with 10²TCID₅₀ SARS-CoV-2 G614 ²¹. While our smaller cohort size may explain this difference in detection of underrepresented animals with lower protection, we detected viral RNA in the lung tissue of two out of four animals vaccinated with Jcovden twice, providing some evidence for initial infection. In any case, vRNA copies in these hamsters’ lungs were significantly lowered, consistent with protection.

Also for Spikevax immunized hamsters, no live virus was found in lungs after infection, before ⁴². Again, viral RNA in lung tissue of mRNA-immunized hamsters was detectable in the Comirnaty- (1/4) or Spikevax-cohorts (3/4).

While these experiments confirmed the VAERD animal model and protection by the authorized vaccines, they also allow direct comparison of the immunogenicity of all four early-approved COVID-19 vaccines. Most interesting, the presence of nAb responses after a single immunization as observed by the AdV vectored vaccines are well reflected by the initial authorization of Jcovden as single-shot vaccine ²²^,51. Consistent with our hamster data, the comparison of humoral immune responses in human vaccinees who received Spikevax, Comirnaty, or Jcovden nevertheless revealed evidence that titers of SARS-CoV-2 binding and neutralizing antibodies induced by a single immunization with Jcovden was significantly lower than the titers observed for the two mRNA vaccines applied in a prime boost schedule ⁵². However, also homologous boosting increases antibody titers in humans immunized with Jcovden ⁵³^,54, reaching just slightly lower titers than individuals who had received two doses of Comirnaty ⁵³, as also found in our experiments, here.

In summary, our hamster study confirms the efficacy of all tested vaccines in the absence of VAERD. Thereby, the rather small differences observed in immunogenicity between the early authorized vaccines go well with observations in vaccinated human populations and further confirm the validity of the Syrian golden hamster model for COVID-19 vaccine research and analysis, but direct transfer between animal model and human patients needs careful analysis, also in the future. Nevertheless, such models are still needed due to constant emergence of new dominant SARS-CoV-2 variants necessitating the adaption of existing vaccines or development of new vaccine concepts over time, which can be tested, in case.

All protocols for animal experiments involving non-human vertebrates, which are described in our manuscript by Ebenig et al., were reviewed, approved, and controlled by the respective competent authority, the Regierungspräsidium Darmstadt, in compliance with the regulations of German animal protection laws and involving the regional animal care committee.

Data Availability Statement

All data generated or analyzed during this study are included in this published article (and its supplementary information files).

Material Availability Statement

Unique biological materials used in this study are available via commercial sources indicated or via the corresponding author of this study upon reasonable request and may require filing of materials transfer agreements (MTAs) and compensation for shipment.

Acknowledgement

The authors would like to thank Carina Kruip for excellent technical assistance, Bevan Sawatsky for support of BSL-3 procedures and the team of the animal husbandry for logistic support with hamster experiments. This work was supported by grants of the German Center for Infection Research (DZIF; TTU 01.805, TTU 01.922_00) and the German Ministry of Health (CHARIS) to M.D.M.

Author Contributions

Conceptualization: MDM

Acquisition of Funding: AK, MDM

Investigation: AE, MVL, MGS, AK, RP, MDM

Visualization: AE, AK, MDM

Supervision: AK, MDM

Materials: TJM

Writing - original draft: AE, MDM

Writing - review & editing: all authors

Declaration of Interest

The authors declare no competing interests

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Hamster Model of Severe COVID-19 Demonstrates Differential Efficacy of first Licensed Western Vaccines in the Absence of Immunopathogenesis

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Abstract

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Introduction

Material and Methods

Cells

Viruses

Syrian golden hamster animal model

Bronchoalveolar lavage (BAL)

Total IgG quantification

Virus neutralization test (VNT)

Determination of lung virus titers in infected animals

RNA preparation

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

Histopathological examination

Hematoxilin-Eosin staining

Sirius Red staining

Results

Discussion

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References

Additional Declarations

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