key: cord-0999920-aexgrojk authors: Mohandas, Sreelekshmy; Yadav, Pragya D.; Shete, Anita; Nyayanit, Dimpal; Jain, Rajlaxmi; Sapkal, Gajanan; Mote, Chandrasekhar title: Protective immunity of the primary SARS-CoV-2 infection reduces disease severity post re-infection with Delta variants in Syrian hamsters date: 2021-11-30 journal: bioRxiv DOI: 10.1101/2021.11.28.470293 sha: c9dddda69fccf686be7e378779ab87df289a6da9 doc_id: 999920 cord_uid: aexgrojk Delta variant has evolved to become dominant SARS-CoV-2 lineage worldwide and there are reports of secondary infections with varying severity in vaccinated and unvaccinated naturally recovered COVID-19 patients. As the protective immunity following the infection wanes within few months, studies of re-infection after prolonged duration is needed. Hence we assessed the potential of re-infection by Delta, Delta AY.1 and B.1 in COVID-19 recovered hamsters after 3 months of infection. Re-infection with Delta and B.1 variants in hamsters showed reduced viral shedding, lung pathology and lung viral load, whereas the upper respiratory tract viral load remained similar to that of first infection. The reduction in viral load and lung pathology after re-infection with Delta AY.1 variant was not marked. Further we assessed the disease characteristics of Delta AY.1 to understand whether it has any replication advantage over Delta variant and B.1 variant, an early isolate in Syrian hamsters. Body weight changes, viral load in respiratory organs, lung pathology, cytokine response and neutralizing antibody response were assessed. Delta AY.1 variant produced milder disease in comparison to Delta variant and the neutralizing response was similar against Delta, B.1 and B.1.351 variant in contrast to Delta or B.1 infected hamsters which showed a significant reduction in neutralization titres against B.1.351. Elevation of IL-6 levels was observed post infection in hamsters after primary infection. The prior infection could not produce sterilizing immunity but the protective effect was evident following reinfection. This indicates the importance of the transmission prevention efforts even after achieving herd immunity. Research in context Evidence before this study Secondary infections with Delta variant are being widely reported and there are reports of increased disease severity. Delta sub lineages with K417N substitution has caused concern worldwide due to the presence of the same substitution in Beta variant, a Variant of Concern known for its immune evasion. The information on the biological characteristics of this sub lineage is also scanty. Added value of this study The present study showed that the secondary infection with Delta variant does not show any evidence of increased disease severity in hamster model. Delta AY. 1 variant produces mild disease in Syrian hamsters in contrast to severe disease caused by Delta variant. Delta, B.1 and AY.1 variant infected hamster sera showed comparable cross neutralizing response against each other. In contrast to the lower neutralizing response shown by B.1 and Delta variant infected animals against B.1.351 variant, Delta AY.1 showed comparable response as that with other variants. Implications of the available evidence SARS-CoV-2 infections do not produce sterilizing immunity but protect from developing severe disease in case of Delta variant re-infection indicating the importance of the transmission prevention efforts even after achieving herd immunity. Delta AY. 1 infection in hamsters did not show any evidence of speculated immune evasion. The information on the biological characteristics of these sub lineages including transmissibility, disease severity and immune evasion are still unknown. SARS-CoV-2 generates neutralising antibody response after infection in humans, but protective immune titre to prevent a subsequent infection is not yet understood. 4 In case of other human coronaviruses (HCoV), waning of immunity is observed in 1 to 3 years and reinfection events have been reported as a common feature of HCoV-NL63, HCoV-229E, HCoV-OC43 and HCoV-HKU1. 5, 6 After the natural SARS-CoV-2 human infection, immune response is suspected to persist for about 90 days in most patients. 4 SARS-CoV-2 re-infection cases with varied disease severity have been published from many countries. 7, 8, 9, 10, 11 The speculated reasons for re-infection are infection with a higher virus dose/another virulent 5 strain, antibody-dependent enhancement and waning of immune response. 8 Laboratory studies have shown that the duration of infection-acquired immunity is inconsistent and the response against variants of concern also differ. 12 The risk of re-infection also depends on host susceptibility, vaccination status, and exposure to COVID-19 patients during infectious phase. 13 Understanding the potential risk of a re-infection is important in improving COVID-19 prevention and control measures. Re-infection studies in the population are very less and the rate of re-infection is also not clear. 14 There are few reports of aggravated disease severity in case of Delta variant re-infection. 15, 16 The impact of the immunity on threat of re-infection posed by different variants still needs to be understood. Such studies are important in making policy decisions which rely on the herd immunity like vaccination. Animal models are important in understanding virus properties, disease pathogenesis, measuring efficacy of countermeasures etc. Syrian hamsters have been widely used in studying SARS-CoV-2 disease characteristics. Our previous studies have shown the pathogenicity and immune evasive properties of the Delta variant in hamsters. 17 Few animal model studies also indicate the neutralizing antibody response generated after primary SARS-CoV-2 infection can reduce the viral load and severity of a second infection. 18, 19 The degree of protection against a secondary infection by Delta lineage variants is still not clear. The ethical approval for the study was received from Institutional Animal Ethics Committee The experiments were performed in the Containment Facility of ICMR-National Institute of Virology, Pune. For the re-infection study, 12 female hamsters, 16-18 weeks old which were previously infected with B.1 variant of SARS-CoV-2 (with an infectious dose of 10 4.5 TCID50) after 3 months of initial infection were used (Figure 1a ). IgG response and neutralizing antibody levels were checked and animals were divided into 3 groups randomly. 7 The hamsters were divided into 4 animal per group and were re-infected with Delta/Delta AY.1/B.1variants with a virus dose of 10 5 TCID50. Swab samples were collected on 2, 4, 6 DPI and body weight change was monitored till 7 days. The hamsters were sacrificed on 7DPI to collect organ and blood samples. As control for the re-infection study and also to understand the disease characteristics of Delta AY.1, three study groups of 17 female, Syrian hamsters of 12-14 week age each were included in the study to assess pathogenicity and a virus dose of 10 5 TCID50 of Delta/ Delta AY.1/B.1 was used intranasally to inoculate the hamsters (Figure 1b ). Swab samples (n=7) were collected on alternate days during the study period. Hamsters were observed for a period of 14 days for body weight loss and 5 hamsters/group were sacrificed on 3, 7-and 14days post infection (DPI) to collect organs and blood samples. Nasal wash, throat swab and organ tissue samples were used for viral load estimation. Organ samples collected during necropsy were weighed and homogenized in sterile media using beads in a tissue lyser machine (Qiagen, Germany). The lysate was used for RNA extraction using the MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit as per the manufacturer's instructions. Quantitative real-time RT-PCR was performed for the E gene of SARS-CoV-2 using published primers to estimate the genomic viral RNA load and for the N gene of SARS-CoV-2 using published primers to estimate the subgenomic viral RNA load. 20,21 The serum samples were tested for IgG antibodies by an in-house developed ELISA 22 . Briefly, inactivated SARS-CoV-2 antigen/ Vero CCL81 cell lysate coated microtiter plates were blocked with liquid plate sealer. Hamster sera samples diluted 1: 100 were added and 8 incubated for 60 minutes at 37°C. The plates were washed following incubation and 1:3000 dilution of anti-hamster IgG-horse radish peroxidase (Thermoscientific, USA) was added and incubated for 60 minutes. The plates were washed and substrate was added to each well for color development. The reaction was terminated with sulfuric acid and the absorbance was measured at 450 nm using an ELISA reader. The assay was performed in duplicate and the assay cut off was set at an optical density value of 0.2 and positive/negative ratio of 1.5. Plaque Reduction Neutralization test (PRNT) was performed against B.1, Delta, Delta AY.1 and Beta (B.1.351) variants as described earlier 23 . Diluted sera were mixed with virus containing a 50-60 plaque forming units/0.1 ml and the virus-sera mixture was incubated for 60 minutes and added in a tissue culture plate with Vero CCL-81 monolayer. After 60 minutes, the mixture was aspirated and media with 2% carboxymethyl cellulose with 2% fetal bovine serum was added. After an incubation period of 4 days, the media was decanted and amido black staining was performed. The plaques were counted and PRNT50 titers were calculated. ELISA based estimation (Immunotag, USA) was performed to assess the levels of IL-4, IL-6, IL-10, IFN-γ and TNF-α in hamster sera samples as per the manufacturer's instructions. Formalin fixed lung tissue samples were processed using an automated tissue processor and were stained by routine hematoxylin and eosin staining. The samples were coded and were blindly scored. The bronchiolar (degeneration, epithelial loss), alveolar parenchymal (edema, 9 exudation, mononuclear infiltration, emphysema, pneumocyte hyperplasia, septal thickening) and vascular lesions (congestion, haemorrhages, perivascular infiltrations) were graded for severity on a score from 0 to 4. Graph pad Prism version 9.2.0 software was used for the descriptive statistics and statistical analysis. Non parametric Mann Whitney tests were used for the analysis. The p-values less than 0.05 were considered statistically significant. The study sponsor has no role in study design, analysis, interpretation of data, in the writing of the report and in the decision to submit the paper for publication. A mean body weight loss of -9.3 ± 6.5 (mean ± standard deviation) % was observed in Delta infected group (p=0.0248 vs Delta AY.1 and p=0.0082 vs B.1) on 6 DPI whereas the Delta AY.1 and B.1 infected group animals showed a average weight gain of 0.23 ± 5.18 % and 1.4 ± 3.34 % respectively (Fig. 2a) . Anti-SARS CoV-2 IgG response could be observed from day 3 in all infected groups with an mean optical density (OD) ± standard deviation (SD) of 0.65± 0.45, 1.00 ± 0.67, 1.02 ± 0.40 on 3DPI and 1.25 ± 0.55, 0.62± 0.13 and 0.62 ± 0.11 on 14 DPI for Delta AY.1, Delta and B.1 variant respectively (Fig. 2b) . Neutralizing antibodies could also be detected in the animals from 3DPI. The mean ± SD of neutralizing antibody titre on 14 (Fig.2c-2e) . DPI, which became more pronounced by 14 DPI in 3/5 animals infected. The highest lung cumulative score was observed in Delta infected group on 7 DPI. By 7DPI, inflammatory changes became severe in Delta group characterized by severe congestion/haemorrhages, alveolar consolidation, loss of bronchial epithelium, septal thickening, pneumocyte hyperplasia, cellular infiltration in the alveolar interstitial space, peri bronchial and perivascular area. In case of B.1 pneumonic changes became more pronounced by 7 DPI (Fig 3f) . 1 1 In the throat swab and nasal wash samples, gRNA could be detected in a decreasing trend till 12 which were significantly lower in Delta AY.1 group (Fig 4f) . In nasal turbinates, the gRNA and sgRNA could be detected till 14 DPI in all the groups. Delta AY.1 group did not show any significant difference in the gRNA or sgRNA level of nasal turbinates in comparison to other groups (Fig 5a, 5b) . In Delta AY.1 group, a lower mean viral load (mean ± SD = 4.55×10 4 ± 9.0 ×10 4) was observed on 7 DPI in lungs, which showed complete clearance of viral gRNA (p=0.0010 vs Delta) and sgRNA (p=0.0057 vs 1 2 B.1) by 14 DPI. Other organs like brain (1/5), heart (2/5) and large intestine (2/5) of Delta AY.1 infected group showed sgRNA positivity on 3DPI and none from the Delta and B.1 infected group (Fig.5c,5d) . After re-infection, the body weight loss in all the infected groups was minimal irrespective of the variant infected ( Supplementary Fig.1a) Post re-infection, the PRNT50 titres also showed rise in titre with all the variants (Table 1) . The serum cytokine levels did not show any significant difference between re-infected groups and the uninfected control sera. The viral load in throat swab and nasal wash were significantly lesser in the Delta (p=0.0286) and B.1 (p=0.0286) re-infected hamsters on 2, 4 and 6 DPI (Fig 6) . Even though the viral load in AY.1 group also showed reduction, the values were not statistically significant. The viral load in nasal turbinates were comparable in both re-infected and the control group irrespective of the variant infected (Fig 7) . Lungs gRNA level in B.1 infected group was significantly lesser (p=0.0286) in B.1 infected group whereas in case of Delta, a slight reduction was seen and AY.1 group showed comparable viral RNA load. In case of primary infection, grossly diffuse haemorrhages in all the lung lobes were seen in 4/5 naïve animals infected with Delta, haemorrhagic foci in one or two lobes in 2/5 animals infected with Delta 1 3 AY.1 and few focal haemorrhages in case of B.1 infection (Fig. 8a-8l) . In contrary the reinfected animal showed only few focal haemorrhagic foci in case of all variant infections ( Fig. 8m-8x) . The lungs body weight ratio after the Delta variant re-infection was reduced in comparison to the primary infected animals (Fig.8y) . In the re-infected Delta and B.1 group, the lung pathological changes observed on 7DPI were milder in comparison to the naïve infected animals. In case of AY.1 re-infection, the re-infected group showed similar disease severity as that of 2/5 naive infected animals (Fig 8z) . 19 Even though a reduction was seen in viral shedding, the nasal turbinate viral load remained comparable. The prior infection could not confer sterilizing immunity in the present study also as reported earlier in rhesus macaque and hamster model. 19, 26, 27 These studies were performed within a month post recovery from primary infection in contrast to our study. Experimentally re-infected or vaccinated animals can shed SARS-CoV-2 through upper respiratory tract. 19, 26, 27, 28, 29 In case of Delta AY.1, sgRNA clearance was observed in ¾ animals by 7 DPI but the average viral load remained comparable to that of the primary infection. Although the risk of re-infection appears to be low in humans, there are reports of varying disease severity in re-infected individuals. 9, 11, 15, 16 Wang et al, 2021 has reported 68.8%, 18.8% and 12.5% of similar, worse and mild disease severity in re-infection cases. 11 Severe disease has also been reported with Delta variant re-infection. 15 , 16 We did not observe any aggravation of lung disease in the re-infected animals with any of the variants. The lung histopathology score of Delta AY.1 variant re-infected group showed pneumonic changes of moderate severity in contrast to the mild disease seen in the primary infection group on 7 DPI. When these observations were compared to the overall histopathology score on 3, 7 and 14 DPI after primary infection with Delta AY.1 variant, the severity was comparable. Another indicator of disease severity in hamster model is the body weight loss which was also found minimal here. Many cytokines has been reported to be increased in severe COVID-19 patients and few like IL-6, IL-8, IL-10 and TNF-α are considered as indicators of severe disease. 30, 31 The increased production of the cytokines can lead to cytokine storm and worsening of the disease prognosis. 32 Here we have observed increased IL-6 cytokine levels after primary infection in hamsters with SARS-CoV-2 variants. We found no aggravation in cytokine responses post re-infection. IL-6, IL1beta, and TNF increase has been reported in hamsters infected with SARS-CoV-2. 33 comparable neutralization for the Delta AY.1 and Delta with the sera of naive BBV152 vaccinees, recovered cases with full vaccination and breakthrough cases in comparison to B.1 variant. 35 Although the disease induced by SARS-CoV-2 in hamsters resemble with humans, many immunological parameters in hamsters are yet to be defined. Our study had limitation of small sample size too. 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