Omicron recently emerged sub-lineage BA.5, together with BA.4, caused a 5th wave of coronavirus disease (COVID-19) in South Africa and subsequently emerged as a predominant strain globally due to its high transmissibility. The lethality of BA.5 infection has not been studied in acute hACE2 transgenic (hACE2.Tg) mice models. Here, we investigated tissue-tropism and immuno-pathology induced by BA.5 infection in hACE2.Tg mice. Our data show that intranasal BA.5 infection in hACE2.Tg mice result in attenuated pulmonary infection and pathology with diminished COVID-19-induced clinical and pathological manifestations. BA.5, similar to Omicron (B.1.1.529), infection led to attenuated production of inflammatory cytokines, anti-viral response and effector T cell response as compared to the ancestral strain. Mice recovered from B.1.1.529 infection showed robust protection against BA.5 infection with reduced lung viral load and pathology. Our data provide insights as to why BA.5 infection escapes previous SARS-CoV-2 exposure induced-T cell immunity but may result in milder immuno-pathology and alleviated chances of re-infectivity in Omicron-recovered individuals.

Flow cytometry and intracellular cytokine staining

SCS obtained from spleen and dLN were either surface stained by using fluorescent anti-mouse antibodies in FACS buffer (PBS with 1% FBS) and analysed as previously described (Rizvi et al, 2021 & 2022); or were in-vitro in the presence of RBD (2µg/ml) or its absence (with phorbol 12-myristate13-aceate (PMA; 50 ng/ml; Sigma-Aldrich) and ionomycin (1 µg/ml; Sigma-Aldrich). RBD stimulation was performed for 6 days and was used for intracellular cytokine staining. For PMA + Ionomycin cells were activated for 4 h in the presence of Monensin (#554724 Golgi-Stop, BD Biosciences). All the cells were first washed and blocked with Fc block (anti-mouse CD16/32, Biolegend) at room temperature (RT) for 20 min. Thereafter, cells were used for  surface staining with α-CD3, α-CD4, α-CD8, α-CD11b, α-NK1.1, α-Gr1, α-c-kit, α-γδTCR, α-FcƐr1α for 20 min at RT in dark. Thereafter, cells were fixed in Cytofix and permeabilized with Perm/Wash Buffer using a Fixation Permeabilization solution kit (#554714, BD Biosciences). Permeabilized cells stained intracellular cytokine antibodies by using -IFNγ, α-IL17A, α-IL10 antibodies in a permeabilizing buffer in the dark for 20 min at RT. The acquisition of the flow cytometry data was done on  (Canto II; BD Bioscience) and analysed on FlowJo software (Tree-Star).

Measurement of viral load 

RNA isolated from lung or other organ samples homogenized in Trizol (Invitrogen) was used for viral load estimation. Quantitation of isolated RNA was done and 1 µg of total RNA was then reverse-transcribed to cDNA using the iScript cDNA synthesis kit (Biorad; #1708891) (Roche). 1:5 diluted cDNAs were then used for qPCR by using KAPA SYBR® FAST qPCR Master Mix (5X) Universal Kit (KK4600) on a Fast 7500 Dx real-time PCR system (Applied Biosystems) and the results were analyzed with SDS2.1 software. Briefly, cDNA was used as a template for the CDC-approved commercial reagent for SARS-CoV-2 N gene: 5′-GACCCCAAAATCAGCGAAAT-3′ (Forward), 5′-TCTGGTTACTGCCAGTTGAATCTG-3′ (Reverse). Beta-actin gene was used as an endogenous control and was used for normalization through quantitative RT-PCR. The region of N gene of SARS-CoV-2 starting from 28287 – 29230 was cloned into pGEM®-T-Easy vector (Promega). This clone was linearized using SacII enzyme and in vitro transcribed using the SP6 RNA polymerase (Promega). The transcript was purified and used as a template for generating a standard curve to estimate the copy number of SARS-CoV-2 N RNA as previously described (Rizvi et al. 2022, 2023)

qRT-PCR

For qPCR from splenocytes or lung samples, RNA isolation and cDNA synthesis was done as described above. Diluted cDNAs (1:5) were then used for qPCR by using KAPA SYBR® FAST qPCR Master Mix (5X) Universal Kit (KK4600) on a Fast 7500 Dx real-time PCR system (Applied Biosystems) and the results were analyzed with SDS2.1 software. Beta-actin gene was used as an endogenous control and was used for normalization through quantitative RT-PCR. The relative gene expression was calculated by -ΔΔCt formula as described previously (Roy et al, 2021, Rizvi et al, 2022, 2023). The following primer sets were used:

Il-13 – 5’-CTTAAGGAGCTTATTGAGGAG-3’ 3’- CATTGCAATTGGAGATGTTG-5’;; Oas2 – 5’-TTATAAAATACCGGCAGCTC-3’ 3’-ATTACAGGCCTCTTTTTCTG-5’; Oas3 – 5’-CCAAACTTAAGAGCCTGATG-3’ 3’-GCCTCTCCTCCTTTATATCG-5’; RNasel – 5’-ATACTGTAGGTGATCTGCTG-3’ 3’-AAGTATCTCCTTCATTCCCC-5’; CCCTACTCATAAAAATCACCAG-3’ 3’-TTGGAATAGCATTTCCACAG-5’; Ifitm3– 5’-AAGAATCAAGGAAGAATATGAGG-3’ 3’-GATCCCTAGACTTCACGG-5’; β-Actin- 5’-TTAATTTCTGAATGGCCCAG-3’ 3’-GACCAAAGCCTTCATACATC-5’; Il-17 – 5’-ACGTTTCTCAGCAAACTTAC-3’ 3’-CCCCTTTACACCTTCTTTTC-5’; Ifn-γ – 5’-TGAGTATTGCCAAGTTTGAG-3’ 3’-CTTATTGGGACAATCTCTTCC-5’; hACE2–5’-TCCATTGGTCTTCTGTCACCCG-3’; 3’AGACCATCCACCTCCACTTCTC-5’

Anti-RBD ELISA

Direct ELISA for detecting anti-RBD antibodies was performed from day 7 or day 14 sera samples as previously described (Samal et al., 2019). Briefly, WT-RBD protein (2 µg/ml) was coated overnight in coating buffer in 96 well high-affinity ELISA plates (Nunc) at 4 °C. Next day, 5 % skimmed milk (blocking buffer) were used for blocking, and then sera samples from the infected mice were added to the RBD bound ELISA plates at different dilutions and incubated at room temperature (RT) for 1 h. Next, three washing steps with wash buffer (PBS + 0.05 % tween 20) were performed followed by incubation with anti-mouse IgG antibody detection antibody conjugated with biotinylated (1:1000 dilution) for 1 h. The wells were subsequently washed and incubated further with the enzyme Avidin-HRP (1:10000 dilution) (Sigma) for 30 min at RT. Finally, wells were washed five times and TMB substrate (Thermo Fisher Scientific) 50 µl was incubated in the dark for 15-20 min at RT. The enzyme reaction was stopped by adding 50 µl of 1 N H₂SO₄. OD was recorded at 450nm in a microtiter plate reader.

FLowJo for FACS files.

GraphPad Prism for graph files.

README file