key: cord-0819233-3hmtzgeo
authors: Kumar, Praveen; Perween, Reshma; Shrivastava, Tripti; Singh, Vanshika; Ahmed Parray, Hilal; Singh, Swarandeep; Chiranjivi, Adarsh; Thiruvengadam, Ramachandran; Singh, Savita; Yadav, Naveen; Jakhar, Kamini; Sonar, Sudipta; Mani, Shailendra; Bhattacharyya, Sankar; Sharma, Chandresh; Vishwakarma, Preeti; Khatri, Ritika; Kumar Panchal, Anil; Das, Supratik; Ahmed, Shubbir; Samal, Sweety; Kshetrapal, Pallavi; Bhatnagar, Shinjini; Luthra, Kalpana; Kumar, Rajesh
title: Non-neutralizing SARS CoV-2 directed polyclonal antibodies demonstrate cross-reactivity with the HA glycans of influenza virus
date: 2021-07-29
journal: Int Immunopharmacol
DOI: 10.1016/j.intimp.2021.108020
sha: c0648266a5d4b776146a1d5a672919ba5f2fa380
doc_id: 819233
cord_uid: 3hmtzgeo

The spike protein of the SARS-CoV-2 virus is the foremost target for the designing of vaccines and therapeutic antibodies and also acts as a crucial antigen in the assessment of COVID-19 immune responses. The enveloped viruses; such as SARS-CoV-2, Human Immunodeficiency Virus-1 (HIV-1) and influenza, often hijack host-cell glycosylation pathways and influence pathobiology and immune selection. These glycan motifs can lead to either immune evasion or viral neutralization by the production of cross-reactive antibodies that can lead to antibody-dependent enhancement (ADE) of infection. Potential cross-protection from influenza vaccine has also been reported in COVID-19 infected individuals in several epidemiological studies recently; however, the scientific basis for these observations remains elusive. Herein, we show that the anti-SARS-CoV2 antibody cross-reacts with the Hemagglutinin (HA) protein. This phenomenon is common to both the sera from convalescent SARS-CoV-2 donors and spike immunized mice, although these antibodies were unable to cross-neutralize, suggesting the presence of a non-neutralizing antibody response. Epitope mapping suggests that the cross-reactive antibodies are targeted towards glycan epitopes of the SARS-CoV-2 spike and HA. Overall, our findings address the cross-reactive responses, although non-neutralizing, elicited against RNA viruses and warrant further studies to investigate whether such non-neutralizing antibody responses can contribute to effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or ADE.

The ongoing SARS CoV-2 pandemic is devastating and has spread its grip worldwide. The spread of this disease has brought about a revolution in the field of vaccinology; the early development of diverse vaccines, a number of which are under clinical trials and few of these are available for emergency use [1] . There are only a few studies that have evaluated commonalities in the immune responses elicited against different corona viruses, other common respiratory viruses (influenza or RSV) or similar enveloped RNA viruses like HIV-1 [2, 3] . The tendency to exhibit host-derived glycans is a common feature of class 1 fusion proteins such as SARS-CoV-2 spike, HIV-1 Env glycoprotein (gp160) and Influenza HA [4] .

Such host derived glycan motifs can serve as the basis for antibody mediated cross-reactivity , or provide mechanisms for viral escape. Glycan directed cross-reactive antibodies can have significant implications for viral neutralizing activity, ADCC mediated protection or ADE of infection.

Numerous retrospective studies have shown that influenza vaccines may enhance crossprotection and responsiveness to COVID-19 [5, 6] . However, the mechanisms behind these cross-reactive immune responses and their co-relations are poorly understood. Contrastingly, a few studies have shown that influenza vaccines have no synergistic or divergent effects on heterologous diseases, such as non-influenza respiratory virus infection (rhinovirus and coxsackie/echovirus infection) [7] [8] [9] .

Both coronaviruses and influenza viruses are single-stranded, enveloped RNA viruses and both are nucleoprotein-encapsulated. However, the genomes of these two viruses vary in polarity and segmentation. The Influenza virus consists of 8 single-stranded, negative, viral RNA segments, whereas, SARS-CoV-2 is a single-stranded, non-segmented, positive-sense, RNA virus. The SARS CoV-2 infection involves a series of conformational changes in the spike (S) protein, which leads to membrane fusion following binding to the host receptor. However, this process requires appropriate activation of the spike protein by host proteases. The furin protease site between S1 and S2 subunits of the SARS-CoV-2 S protein is homologous to the highly pathogenic influenza viruses [10] .

The envelope proteins of SARS-CoV2 and influenza have evolved to be extensively glycosylated and these glycans are derived from host-cells. The envelopes of these viruses fuse with the host cell utilizing a Class I fusion mechanism, which does not require any other viral surface proteins for fusion [4] . The glycan shield on these viruses provides diverse structural and functional features which help with the viral life-cycle and immune evasion mechanisms by misdirecting the humoral immune response to target non-neutralizing epitopes. Glycan density is especially high in some of the class I fusion proteins [11] , which is consistent with their role in shielding. Differences in the composition, density, and conservation of glycans have been observed across distinctive families of enveloped viruses, e.g. HIV-1, SARS, influenza virus, Lassa, Zika, dengue, and Ebola viruses [12, 13] . However, cross-reactive responses of newly emerged SARS-CoV-2 antibodies and their potential protective or adverse responses are poorly understood.

In this study, we aimed to investigate the reactivity of the SARS-CoV-2 directed antibodies, present in convalescent donor sera and spike immunized mice sera, and investigated whether they confer cross-reactive protection against influenza virus, in terms of neutralization. Our findings highlight that SARS-CoV-2 specific antibodies in the convalescent sera are crossreactive, although they do not exhibit any potential to cross neutralize, as shown by viral neutralization assays, ELISA and other immune-reactive studies. Further, we characterized the epitopes defining the cross-reactivity among the crucial viral targets studied herein, in an attempt to identify shared epitopes towards immunogen design and therapeutic targets against SARS-CoV-2. We believe our findings will aid in understanding whether the antibodies elicited during natural infection or through active immunization can provide protection against circulating infection or lead to disease enhancement as a long-term response in the event of future re-emergence or co-infections.

A longitudinal cohort of COVID-19 positive patients was enrolled at designated COVID-19 testing centers or hospitals within five days of their positive RT-PCR test. The study protocol was approved by the Institute Ethics Committees of all participating institutions. The eligibility criterion was a confirmed positive RT-PCR test for SARS-CoV-2 using nasopharyngeal swabs.

These patients were enrolled after written consent and baseline active phase samples were collected, processed and archived at the NCR Biotech Science Cluster Biorepository for all subsequent analyses. The follow-up visits were designed to capture the clinical outcomes of illness (10-28 days after being diagnosed with SARS-CoV-2 infection), early (6-10 weeks) and late (6 and 12 months) convalescent periods. The duration of illness was defined as the date of onset of symptoms in symptomatic participants and from the date of testing positive for SARS-CoV-2 infection among those who were asymptomatic.

Recombinant proteins were expressed using the Expi 293 F expression system, from a codonoptimized nucleic sequence of RBD-His, Spike-His, HA-His, following the methodology published earlier [14] [15] [16] . Briefly, the culture supernatant was harvested 5-7 days posttransfection and purified by Ni-NTA affinity chromatography followed by dialysis in phosphate buffer saline (pH 7.4) as described in our previous papers [14] [15] [16] [17] . The N protein of SARS-CoV-2 was expressed in the bacterial expression system and was purified by Ni-NTA affinity chromatography.

Animal immunizations for RBD and spike proteins were performed on 7-8 week old, C57BL/6 (male) mice inbred in the THSTI small animal facility (SAF) with 5 animals immunized in each group based on prime/boost immunization regimen, as described in the the published literature [18] . For influenza HA and SARS-CoV-2 N protein, 6-8 week old BALB/c (male) mice, inbred in THSTI small animal facility (SAF) were immunized (i.m; intramuscular route) with 30 µg of the purified recombinant protein in combination with AddaVax as adjuvant in a prime/boost immunization regimen, prime and boost immunization was done 21 days apart.

Pre-bleed sera was collected on day 0 and sera post immunization were collected 14 days after each immunization. For our studies, we used sera collected 14 days after the booster dose. All experiments were performed in accordance with the Guidelines of the CPCSEA, under the protocol approved by the Institutional Animal Ethics Committee (IAEC Approval number: IAEC/THSTI/53 and IAEC/THSTI/93).

For ELISA binding assays, NUNC Maxisorp plates (Thermo Scientific) were coated with 100 μl of recombinant soluble proteins (RBD protein, bovine serum albumin, soluble S protein, and N -protein 2 µg mL -1 ) overnight in coating buffer, 0.1 M Sodium Carbonate, pH 9.5 at 4ºC. No. M6882).

Purified soluble H1-HA was covalently coupled to Tosylactivated MyOneDynabeads (Life Technologies Inc. Cat. No 65501 ) according to the manufacturer's protocol as described by

Patil et al; 2016 [16] . For depletion studies, SARS-CoV-2 infected human serum (neutralization CPEE titre 32200) was diluted to 1:50 in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). Diluted serum (800 µl) along with magnetic beads were incubated at room temperature for 30-60 mins. Unbound plasma antibodies were separated from bound antibodies to protein-coated beads using a DynaMag 15 magnet as described above. This step was repeated 5 times for the depletion of serum antibodies. As a negative control, serum antibodies were depleted from BSA coated beads in parallel.

Purified soluble H1-HA was covalently coupled to Tosylactivated MyOneDynabeads (Life Technologies Inc.) according to the manufacturer's protocol as described by Patil et al., 2016 [16] . In the mice study, HA-coated beads were incubated for 1 h at RT with the sera from HA, spike, and SARS-CoV-2 nucleoprotein immunized groups. For control, pre-immune mice sera and beads with a secondary antibody were included. The beads were incubated with antimouse secondary antibody labelled with Alexa Fluor 488 (Jackson ImmunoResearch, Code:

115-545-062), post two washes with 1% FACS buffer. Beads were finally washed twice and re-suspended in 1% FACS buffer and data were acquired by a BD FACS Canto-II flowcytometer.

For the human study, HA-coated beads were incubated with sera from SARS-CoV-2 positive patients for 1 h at RT. For control, beads were added with secondary antibody only. The staining procedures followed were the same as described above. The secondary antibody used was anti-human PE (Jackson ImmunoResearch, Code:109-116-097 ). Flow data were analysed using FlowJo software and statistical analysis was done by applying 't-test' using GraphPad Prism 7 software.

Protein de-glycosylation was performed by PNGase F (NEB, Cat. No P0705S) (Non-Denaturing Reaction Conditions) and Endo H (NEB, Cat. No P0702S) following the manufacturer's protocol. Briefly, 20 µg of purified dialyzed influenza HA and SARS-CoV-2 spike protein was mixed with Glyco Buffer 2 in 20 µL volume of the reaction mixture. 2 µL of PNGase F was added to the final reaction and reaction mixture was incubated at 37°C for 4 h.

As a control, to estimate the extent of de-glycosylation, one protein reaction with Glyco Buffer 2 and without PNGase F was also incubated at 37°C for the same time period. Both control and enzymatic reaction samples were further run on SDS-PAGE and the extent of deglycosylation was estimated by the shift in mobility of protein bands.

For de-glycosylation, using Endo H, 20 µg of proteins was mixed with Glycoprotein Denaturing Buffer in 10µl of reaction volume and incubated at 100°C for 10 mins. The denatured protein was further mixed with Glyco Buffer 3 and 2 µL of Endo H and reaction mix was incubated at 37°C for 2 h.

CPE based neutralization assays were performed as described previously in Parray et al., 2020 [14] . Briefly, 1 × 10 2 TCID 50 isolate; USA-WA1/2020 virus, passaged once in Vero cells, was incubated with serum dilution ranging from 1:20 to 3260 for 90 mins, followed by 1 h of adsorption on the Vero cells. After washing the cells, DMEM supplemented with 2% (vol/vol) FBS was added. The presence of cytopathic effect (CPE) in cells was detected using a microscope after incubation for 4-5 days at 37°C with 5% CO2. Non-infected VERO E6 cells were used as a positive control, and infected VERO E6 cells were used as a negative control. 

As both SARS-CoV-2 and the influenza virus show similar clinical presentations, we first investigated any possible correlation between the two diseases in terms of reactivity or crossreactivity. We selected SARS-CoV-2 polyclonal sera obtained from SARS-CoV-2 infected human subjects with high titres of neutralizing antibodies (cytopathogenic effect value, CPE 3200), to evaluate them for cross binding to influenza HA proteins by ELISA. Interestingly, the SARS-CoV-2 human polyclonal sera showed a varied degree of cross-reactivity with influenza HA protein (Fig 1A (I) (II)). The cross-reactivity of the polyclonal sera was further confirmed by western blot analysis. Detection of ~70kDa band for HA protein substantiate the cross-reactive binding of the SARS-CoV-2 polyclonal sera with HA proteins (Fig. 1B) .

To further confirm the above results, we investigated the binding of the immune sera, from SARS-CoV-2 spike protein (prefusion spike trimer S2P) immunized mice, and cross-reactivity with influenza HA proteins (Fig1C (I) (II)). The hyperimmune spike sera from mice also showed a similar cross-reactive binding pattern to influenza HA protein both in ELISA and western blot analysis (Fig1D) . To test the specificity of this cross-reactivity and rule out any pre-existing cross-reactive antibodies in mouse polyclonal sera, pre-immune sera sera from mice and sera from mice immunized with the SARS-CoV-2 nucleoprotein (N) were used as a negative control. Neither the pre-immune sera nor the sera from the N-immunized mice showed any reactive binding to the tested proteins (Fig1C.).

The above findings were confirmed by a flow cytometry-based assay in which purified soluble HA protein was coated on magnetic beads and tested for reactivity against SARS-CoV-2 convalescent sera and spike protein immunized mice sera. Similar to the ELISA results, we found that the HA coated beads produced significant positive signals with both human and mice immunized sera when compared to sera from N immunized mice (used as control), which did not show any reactivity with the coated beads ( Fig. 2A I, II, III, IV & Fig. S1 ). infected human serum, no staining was observed, serving as an experimental negative control (Fig.2B) . Our data suggests that this cross-reactivity of anti-SARS-CoV-2 human sera or hyperimmune mice sera is due to the antibody responses directed towards the spike protein of SARS-CoV-2 and the possible presence of shared epitopes between SARS-CoV-2 and the Influenza A virus.

Next, we investigated whether these anti-SARS-CoV-2 cross-reactive antibodies confer any cross-neutralization. To validate the presence or absence of cross-neutralization potential of the cross-reactive anti-SARS-CoV-2 antibodies, we performed serum depletion assays, where HA cross-reactive binding antibodies were depleted from the convalescent serum of a SARS-CoV-2 infected donor that demonstrated potent anti-SARS-CoV-2 neutralizing activity ( Fig   2C) . Depletion was performed using Dynabeads coated with purified H1-HA protein. The depletion of SARS-CoV-2 infected human serum of antibodies directed at HA trimer protein did not show any change in neutralization potency of the polyclonal antibodies towards the SARS-CoV-2 virus, suggesting that cross-reactive binding antibodies to HA proteins do not confer cross-neutralization ( Fig 2D) .

We further investigated the possible bidirectional cross-reactivity of influenza HA immunized sera with SARS-CoV-2 proteins. The mice were immunized with the purified H1-HA protein.

We observed that the HA hyperimmune sera showed cross-reactive binding towards the spike protein of SARS-CoV-2 both in ELISA (Fig 3A (I) (II) ) and in a Western blot (~180kDa band size) (Fig 3B) . Interestingly, HA polyclonal antibodies did not cross-react with the receptor binding domain (RBD) or the N protein of SARS-CoV-2 as compared to the spike in the ELISA binding assay. These findings support the hypothesis that antibodies in the sera of HAimmunized mice target non-RBD epitopes in the spike protein. The hyperimmune sera of N protein of SARS-CoV-2 immunized mice was and sera of pre-immunized mice showed no reactivity to the full length full length spike or RBD proteins. Our experimental findings show that the cross-reactivity in binding is common between SARS-CoV-2 and HA immunized sera and is bidirectional, with no reactivity towards other proteins tested in the present experimental setup, suggesting some commonality of the epitopes between the HA and SARS-CoV-2 spike protein, excluding the RBD region.

We next sought to determine the possible cross-neutralization capabilities of these crossreactive antibodies. HA immunized hyperimmune sera were tested against SARS-CoV-2 virus in a CPE based virus neutralization assay. None of the tested sera showed any reduction in cytopathic effect at 1:20 serum dilution (Fig 3C) .

To confirm and investigate the epitopes shared by SARS-CoV-2 and influenza HA proteins, which directing the cross-reactivity among the two group of proteins, we studied the possible contribution of glycosylation. We found that deglycosylation of HA protein shows selectively decreased reactivity towards SARS-CoV-2 polyclonal antibodies in a western blot (Fig. 4A) .

Whereas the fully glycosylated HA Envs bind better with the SARS-CoV-2 polyclonal sera than PNGase H deglycosylated Envs. In a similar manner, the spike protein was deglycosylated and tested for HA immune sera cross-reactivity. The HA immune sera showed reduced binding reactivity towards the PNGase H deglycosylated spike protein (Fig. 5A) . This data suggests that cross-reactive binding antibodies present in HA and spike immune sera show bi-directional reactivity and are targeted towards glycans.

To further elucidate the role and the extent of involvement of complex glycosylations in crossreactivity, we treated both the HA and spike protein with the Endo H enzyme; that selectively removes high mannose and hybrid glycans, and tested for its bi-directional cross-reactivity.

The immune sera shows minimumal difference in the recognition of Endo H treated and untreated proteins in the western blots, the Endo H treated HA protein shows significantly less changes in the recognition of spike sera as compared to treated spike protein, which shows slightly reduced reactivity (30%) with the HA sera ( Fig.4B & 5B ). However, when tested in a methyl α-d-mannopyranoside (a stabilized mannose analogue) ELISA-based competition assay for the cross-reactive interactions, both the protein shows partial disruption of the binding with the sera at the highest concentrations of methyl-d-mannopyranoside (750 mM), indicating that these interactions are moderately susceptible (~35-40 % inhibition) in the presence of mannose analogue (Fig 6A & B) . Hence, our results suggest that the cross-reactive antibody responses are preferentially directed towards N-linked complex type glycans with differential involvement of complex glycosylation.

To investigate if any specific IgG subclasses are contributing to driving the mechanism of cross-reactive response, we evaluated the antibody isotypes and specificities of HA and spike hyperimmune sera against the cross-reactive protein. The IgG1 subclass was found to be the most dominant class of cross-reactive antibodies. The H1-HA mice polyclonal sera cross-reacts with spike protein and the most dominant class of cross-reactive antibodies were IgG1 ( Fig   6C) . However, in spike immunized mice sera that cross-reactive antibody isotype with HA proteins was IgG1 followed by IgG2b, IgG2c and IgG3 (Fig 6D) . It will be important to study the antibody isotypes and subclasses of these cross-reactive antibodies generated in response to SARS-CoV-2 in the natural course of infection and their role in COVID-19 pathogenesis.

Several recent large-cohort studies have shown that receiving an influenza vaccine shot before or shortly after contracting SARS-CoV-2 improved health outcomes and reduced the risk of contracting a severe COVID-19 infection [5, 19, 20] . Our study shows that non-neutralizing SARS-CoV-2 directed polyclonal antibodies (SARS-CoV-2 convalescent sera and spike immunized mice sera) demonstrate cross-reactivity with the HA glycans of influenza virus.

However, there is no scientific report so far that elucidates the mechanism behind this possible cross-protection [21] . Our study describes that antibodies elicited against influenza HA protein; a major component of the flu vaccine, cross-react with the spike protein of SARS-CoV-2, though these cross-reactive antibodies do not show any direct protection in terms of neutralization of the SARS-CoV-2 virus. Evaluation of the ADCC activity of these antibodies will reveal if their effector functions can confer any protection, though it has not been addressed herein and is a limitation of the study. Using targeted antibody-depletion experiments, we demonstrated that SARS-CoV-2 antibodies that cross-react with HA antibodies are preferentially non-neutralizing to SARS-CoV-2. We found that these cross-reactive antibodies do not bind or bind poorly to the RBD protein. One possible reason for the non-neutralizing behaviour of these cross-reactive antibodies could be that these antibodies target non-RBD regions, as RBD is a highly immunogenic component of spike protein and is recognised by the majority of nAbs, and is, therefore, a major target of current nAb-based vaccine design efforts [22, 23] . Another possible explanation for the poor binding of these cross-reactive antibodies to the SARS-CoV-2 RBD could be the limited number of glycosylation moieties within the RBD. Any protection that these cross-reactive antibodies may confer, possibly by enhancing innate immune functions by binding to FcRs, including ADCC and complement activation, might help in the clearance of the virus and reduce the severity of the disease. In a study conducted by Zanettini et al, it was observed that the influenza vaccine had a positive effect on COVID-19 mortality in the elderly population and is supportive of our findings [24] . Viral envelope spike and HA proteins are heavily glycosylated with a variable array of hostderived glycans [25] . These glycans play an important role in viral defence mechanisms via epitope occlusion and host immune system evasion [26] . These glycans are immunogenic in nature and are reported to elicit potent neutralizing antibodies in the case of HIV-1 (2G12) [27] and SARS-CoV-2 spike protein (S309) [28] . In SARS-CoV-2, glycosylation of the spike protein is essential for viral infection and is important for viral escape and defence mechanisms [29] . The shedding of viral glycoproteins can redirect the humoral immune response by exposing immunodominant (non-neutralizing) epitopes, not exposed on the functional native closed trimeric conformation of the Env proteins, which plausibly leads to the production of cross-reactive binding antibodies which are, however, non-neutralizing [3, 30] .

reactive antibody responses towards the FP and HR2 epitopes of endemic CoVs, implying that B cell memory for these epitopes exists in the general population and hypothesised that these antibodies might exhibit cross neutralisation to SARS-CoV-2 [31, 32] . Several epidemiological studies have indicated a substantially higher prevalence of cross-reactive antibodies against SARS-CoV-2 that protect against SARS-CoV-2 infection in sub-Saharan African populations, with a much lower COVID-19-related morbidity and mortality rate, plausibly due to these regions having a higher incidence rate of infectious diseases [33] . In a similar study, it has recently been documented that a large fraction of non-exposed individuals show antibody cross-reactivity and T-cell reactivity to SARS-CoV-2 peptides. Such antibodies, which developed during the pre-pandemic period, are probably those elicited against homologous peptides shared by the endemic HCoVs and related viruses [34, 35] . Future studies need to address the functional implications of these cross-reactive antibody responses to understand how the history of an individual's exposure to the endemic virus can influence their immune response to COVID-19 infection and disease progression.

The findings of our study suggest that most of these cross-reactive antibodies target the glycoepitopes (influenza HA). In dengue infection, it has been shown that human or hamster polyclonal cross-reactive antibody response against West Nile virus (WNV) is primarily directed against a cross-reactive domain II fusion loop epitope (DII-FL) on the envelope (E) protein; these cross-reactive antibodies were found to be poorly neutralizing. Passive transfer of these purified cross-reactive IgGs from DENV-immunized hamsters protects mice from lethal WNV infection via Fc receptor and complement-dependent effector mechanisms [36] .

Furthermore, we discovered that HA immunized mice's sera did not cross-react with the RBD of SARS-CoV-2 and could not neutralize the Wuhan live SARS-CoV-2 virus in a CPE assay.

The background signal in SARS-CoV-2 serological assays, leading to a false predictive value between antibody titers and viral neutralisation, disease status, and disease progression, may be attributed, at least partially, to such cross-reactive antibodies. Our results suggest that adding RBD and/or N protein to similar serological assay platforms, with no cross-reactivity, may reduce the number of false positives, improve sensitivity and may be useful signatures for differentiating vaccine responses.

In our analysis, we found that SARS-CoV-2 and HA displayed bi-directional and similar mechanistic (glycan-dependent) cross-reactivity, possibly since both these viruses share the same ecological niche and exhibit a similar mode of propagation and clinical presentation [37, 38] . One possible explanation for the emergence of cross-reactive non-neutralizing antibodies might be deceptive imprinting of the immune system towards non-protecting epitopes [39] and the mechanism that favours viral escape through pre-existing cross-reactivity.

The immune response, though cross-reactive with the evading virus is unable to mount a protective neutralizing response. These cross-reactive antibodies can form immune complexes with the virus and can activate systemic immune responses that can indirectly help with viral degradation pathways [40, 41] . Nucleoprotein (N) protein of SARS-CoV-2 immunized mice sera and pre-immune sera was used as experimental negative control. The binding and immunoblot assays were repeated at least three times.

Data are presented as the mean ±SD, and differences between groups were determined by two-way analysis of variance (ANOVA) followed by Tukey's post hoc tests using GraphPad Prism 7. Statistical significance between the control and different groups is shown as *P < 0.05, **P < 0.01, ***P < 0.0002, ****P < 0.0001. Nucleoprotein (N) protein of SARS-CoV-2 immunized mice sera and pre-immune sera were used as an experimental negative control. C. Cross neutralization potential of sera was tested in CPE based assay.

The value represents the 100% serum neutralization titers. The neutralization assay was done in duplicate and repeated at least three times. Statistical significance between the control and other experimental groups was estimated by two-way analysis of variance (ANOVA) followed by Tukey's post hoc tests using GraphPad Prism 7. Statistical significance between control and different groups is shown as *P < 0.05, **P < 0.01, ***P < 0.0002, ****P < 0.0001. and poorly reacts with PNGase F deglycosylated protein. The immunoblot assay was repeated at least two times. As a control experiment same blot/in parrel, equal amount of both proteins were loaded and probed with Anti-His tag antibody, (B). In this, the spike protein was de-glycosylated with the endo H enzyme and Western blot analysis was performed with H1-HA immunized mice sera. The H1-HA mice bind with both the glycosylated and endo H deglycosylated proteins. The immunoblot assay was repeated at least two times. As a control experiment same blot/in parrel, equal amount of both proteins were loaded and probed with Anti-His tag antibody. The relative expression/decrease in binding is calculated by densitometry analysis and is represented in bottom panel as bar diagram. Statistical significance was determined using t-test and p<0.05 was considered significant. 

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Antibody binding was quantified via ELISA. All the experiments were performed three times separately. Data are presented as the mean ±SD, and differences between groups were determined by two-way analysis of variance (ANOVA) followed by Dunnett post test using GraphPad Prism 7. Statistical significance between control and different groups is shown as P>0.05 (N.S not significant), *P < 0.05, **P < 0.01, and ***P < 0.0001.C & D. Isotyping of crossreactive antibodies in the mice immune sera was tested in ELISA binding assay

Funding: This work was supported through the THSTI core grant.

The authors declare that they have no conflict of interest