key: cord-1045420-05505vvv
authors: Bhuiyan, Taufiqur Rahman; Akhtar, Marjahan; Khaton, Fatema; Rahman, Sadia Isfat Ara; Ferdous, Jannatul; Alamgir, A S M; Rahman, Mahbubur; Kawser, Zannat; Hasan, Imrul; Calderwood, Stephen B; Harris, Jason B; Charles, Richelle C; LaRocque, Regina C; Ryan, Edward T.; Banu, Sayera; Shirin, Tahmina; Qadri, Firdausi
title: Covishield vaccine induces robust immune responses in Bangladeshi adults
date: 2022-04-29
journal: IJID Regions
DOI: 10.1016/j.ijregi.2022.04.006
sha: ba0c1a1c1bfbd45d49fbc086c7b579f1b369b462
doc_id: 1045420
cord_uid: 05505vvv

Objectives : SARS-CoV-2 specific antibody responses after Covishield vaccination has been evaluated until 6 months after vaccination. Design : We assessed SARS-CoV-2 specific antibody responses by ELISA to recombinant receptor-binding domain of SARS-CoV-2 in 381 adults given the Covishield vaccine at baseline (n=119), 1 month (n=126) and 2 months (n=75) after the 1st dose, 1 month after the 2nd dose (n=161) and monthly for 3 additional months. Results : Over 51% of the vaccinees were seropositive at baseline before vaccination with Covishield and almost all participants (159/161) became seropositive 1 month after 2nd dose. The levels of antibodies peaked at 1 month after receipt of the second dose and then subsequently decreased by 4 months after the first dose; the lowest responses were found at 6 months after first vaccine dose, although significantly higher antibody responses and responder frequencies remained (P<0.0001) compared to baseline. Compared to younger participants, older participants had lower levels (P<0.05) of antibody responses 6 months after first vaccination. Participants who had previous infection with SARS-CoV-2 showed a robust higher antibody response after vaccination. Conclusions : These findings help elucidate longevity of vaccine-specific antibody responses following Covishield and provide information relevant to planning boosting after initial two dose vaccination.

Objectives: SARS-CoV-2 specific antibody responses after Covishield vaccination has been evaluated until 6 months after vaccination.

Design: We assessed SARS-CoV-2 specific antibody responses by ELISA to recombinant receptor-binding domain of SARS-CoV-2 in 381 adults given the Covishield vaccine at baseline (n=119), 1 month (n=126) and 2 months (n=75) after the 1 st dose, 1 month after the 2 nd dose (n=161) and monthly for 3 additional months.

Results: Over 51% of the vaccinees were seropositive at baseline before vaccination with Covishield and almost all participants (159/161) became seropositive 1 month after 2 nd dose. The levels of antibodies peaked at 1 month after receipt of the second dose and then subsequently decreased by 4 months after the first dose; the lowest responses were found at 6 months after first vaccine dose, although significantly higher antibody responses and responder frequencies remained (P<0.0001) compared to baseline. Compared to younger participants, older participants had lower levels (P<0.05) of antibody responses 6 months after first vaccination. Participants who had previous infection with SARS-CoV-2 showed a robust higher antibody response after vaccination.

Conclusions: These findings help elucidate longevity of vaccine-specific antibody responses following Covishield and provide information relevant to planning boosting after initial two dose vaccination.

Keywords: Covishield, seropositive, antibodies, Bangladesh, comorbidity

The SARS-CoV-2 pandemic caused more than 300 million confirmed COVID-19 cases and about 5.5 million deaths globally by the end of January 2022 (worldometer, 2022) . There is a desperate need for widely available and effective vaccines against Coronavirus Disease 2019 (COVID-19) (Dhama et al., 2020) . Currently, there are at least 216 COVID-19 vaccine candidates in development, including 92 ongoing clinical trials (bioRENDER, 2022). Bangladesh has 7 vaccines for COVD-19 under emergency use authorization.

In Bangladesh, the COVID-19 vaccination program was initiated on January 27, 2021, and mass vaccination started on February 7, 2021 for all adults who are above 40 years, including front line health workers in any age group above 18 years; this program was expanded to include school age children (12-17 years) beginning in November, 2021 (Mon Nov 1, 2021 . For this program, the government of Bangladesh purchased 30 million vaccine doses (e.g: Covishield, Comirnaty, Spikevax, BBIBP-CorV) for the Bangladeshi population.

The adenovirus vector ChAdOx1 to deliver a COVID-19 vaccine (Covishield; Oxford/ AstraZeneca COVID-19 AZD1222; Serum Institute of India)) has EUA for use in Bangladesh as well as in several other countries globally, including the UK, the EU, Argentina, Brazil, Dominican Republic, El Salvador, India, Malaysia, Mexico, Nepal, Pakistan, the Philippines, Sri Lanka, and Taiwan, and studies have shown that the vaccine is safe and effective (Knoll and Wonodi, 2021, Voysey et al., 2021) . A test-negative, case-control study conducted in India during the delta variant surge showed that the effectiveness of two doses of Covishield against moderate-to-severe COVID-19 disease was 81.5 % (95% CI 9·9-99·0 ) (Thiruvengadam et al., 2021) . Moreover, fully vaccinated people were highly protected against severe infection, hospitalization and death caused by the virus. Covishield appears to be immunogenic across all age groups above 18 years. After vaccinaton, antibody responses against the SARS-CoV-2 spike protein were induced in adults (Ramasamy et al., 2021) . Studies show that history of COVID-19 positivity impacts the magnitude and quality of antibody response after a COVID-19 vaccination; prior infection resulted in quicker and more robust vaccine-induced immune responses compared to participants who were infection-naïve (Tut et al., 2021) . However, in most studies immune responses were measured at only one or two time points after vaccination, hence the impact of prior infection on overall kinetics and longevity of the responses are not known. We report here on the antibody response following Covishield vaccination in 381 Bangladeshi adults who were followed for 6 months. We measured IgG antibodies to the recombinant receptor binding domain (RBD) of SARS-CoV-2 before vaccination and followed the kinetics of antibody responses longitudinally in participants, examining for differences in responses by age, gender, co-morbidities and prior COVID-19 infection.

The study was conducted at the icddr,b (International Centre for Diarrhoeal Disease Research, 

Blood samples were collected from enrolled participants at 7 different time points: day 0 (n=119, before 1 st dose of Covishield vaccination), day 30±10 (n=126, 1 month after the first dose), day 60±10 (n=75, 2 months after the 1 st dose, before 2 nd dose of Covishield vaccination), day 90±10 (n=161, 3 months after the 1 st dose and 1 month after 2 nd dose), day 120±10 (n=32, 4 months after the first dose and 2 months after 2 nd dose ), day 150±10 (n=57, 5 months after the first dose and 3 months after 2 nd dose) and at day 180±10 (n=47, 6 months after the first dose and 4 months after 2 nd dose). Serum was separated from blood after centrifugation at 700x g for 15 minutes and then frozen (-80 O C) until the time of laboratory analysis.

We determined the concentration of RBD-specific IgG antibody by ELISA as described previously using an IgG-specific anti-RBD monoclonal antibody (Akter et al., 2022 , Bhuiyan et al., 2022 , Shirin et al., 2020 , Taufiqur Rahman Bhuiyan, 2022 . The RBD antigen of the spike protein of SARS-CoV-2 and the anti-RBD monoclonal antibody were gifts from A. Schmidt lab, Ragon Institute, Boston MA) (Iyer et al., 2020) . Briefly, 96 wells Nunc® MaxiSorp™ plates (ThermoFisher) were coated with 100 µL of SARS-CoV-2 RBD antigen (1 μg/ml in carbonate buffer) and incubated for an hour at room temperature (RT). After 30 minutes blocking at RT with 5% nonfat milk, heat-inactivated serially diluted serum samples were added to the plate as described previously (Akter et al., 2022 , Shirin et al., 2020 and incubated for an hour at 37°C.

Finally, peroxidase-conjugated secondary antibodies (Goat anti-human IgG, Jackson ImmunoResearch) were added to plates and incubated at ambient temperature for 30 min followed by 5 washes with PBST and one wash with 1X PBS. Bound secondary antibodies were detected using ortho phenylenediamine (OPD; Sigma) and 30% H 2 O 2 after 20 minutes incubation at RT in the dark. Optical density (OD) was measured at 450 nm and 570 nm in the Eon (Biotek) ELISA Reader; OD values were adjusted by subtracting 570 nm OD nm from the 450 nm OD. Based on pre-pandemic serum specimens collected from healthy individuals as well as sera obtained from influenza patients, as discussed previously (Akter et al., 2022 , Shirin et al., 2020 we determined 500 ng/ml (0.5 µg/ml) as the cut-off value for IgG seropositivity.

SARS-CoV-2 IgG specific antibody responses in Bangladeshi vaccinated participants before and after Covishield vaccination were analyzed in this study. Statistical analysis was performed using the Mann-Whitney U test. Graph Pad Prism (version 6.0) was used for generating plots and analyses.

381 healthy adults who received the Covishield vaccine in Dhaka, Bangladesh were enrolled in the study. 124 (33%) of the participants were below 40 years of age, all of whom were eligible for vaccination as front liner workers who performed COVID-19 related work. and 257 (67%) participants were 40 years and above (Table 1) . More male (64%, n=244) participants entered the study. Based on self-reported history prior to vaccination, 40% (n=152) of participants had laboratory-confirmed COVID-19 infection (COV-P) in the three to ten months before receiving the first vaccine dose. Among the participants, 22% (n=84) did not elect to provide their comorbidity history. Of the remaining participants, 38% (n=144) had a co-morbid condition (e.g. asthma, diabetes, heart diseases, Kidney disease, hypothyroidism and cancer, Table 1 ).

Two doses of the Covishield vaccine elicited strong antibody responses at all time points, and these decreased but remained significantly elevated out to 6 months following the first vaccine dose (Fig 1) . Before vaccination, 51% of the participants already had SARS-CoV-2 antibodies over 500 ng/ml (cut-off concentration for seropositivity). Overall, at baseline, the geometric mean concentration of SARS-CoV-2 IgG was 403 ng/ml. One month after the first vaccine dose, 92% of vaccinees were seropositive and the responses were significantly higher (geometric mean 2727 ng/ml) compared to pre-vaccination level (P<0.0001). The level of IgG antibodies increased even further (geometric mean 4029 ng/ml) after 2 months, before the 2 nd dose of the Covishield vaccine (Fig 1) . One month after the 2 nd dose (3 months after the first dose), a strong response (geometric mean 8217 ng/ml) was seen and almost 99% of the vaccinees had RBD-IgG antibodies and at this time point, the highest magnitude of IgG antibody response was observed.

Two months after the second dose (4 months after the first vaccination), 100% of the vaccinees had seropositive levels of IgG antibody above the 500 ng/ml cut-off, and these remained similar until 6 months after the first vaccination. Compared to baseline, IgG antibody responses remained significantly (P<0.0001) elevated out to 6 months following initial vaccination, however, the antibody concentration had decreased (geometric mean 3271 ng/ml) by 6 months (P<0.0001) compared to the level observed 3 months following vaccination (Fig 1) .

Since many Bangladeshi participants had already been infected with SARS-CoV-2 prior to vaccination, we compared antibody responses between vaccinees who had no history of COVID-19 positivity (COVID-19 negative, COV-N) and those who were previously COVID-19 positive (COV-P) by RT-PCR using nasopharyngeal swab specimens (Fig 2A) . Covishield vaccine elicited strong IgG antibody responses in the both COV-N and COV-P groups. In the COV-N group, 35% of participants were seropositive for IgG at baseline (geometric mean 256 ng/ml). By one month following the initial vaccination, 90% of the vaccinees without a prior diagnosis of COVID became seropositive and almost all became seropositive 2 months after receiving the second dose. Compared to baseline, significantly (P<0.0001) higher IgG antibody responses were found at all time points out to 6 months following the first vaccination.

In contrast, 70% of the prior COV-P participants were seropositive before the first dose of the Covishield vaccine but their antibody titers significantly increased further after getting both doses of the vaccine (Fig 2A) . At baseline, the IgG level was significantly higher (P<0.001, 2.7 fold higher geometric mean, Fig 2B) in COV-P compared to COV-N participants. After receiving the first and second dose of the Covishield vaccine, IgG concentrations were significantly higher (P<0.05 to P<0.0001) in the COV-P group at all time points out to 3 months (one month after 2 nd dose). Before giving the second dose, that is 2 months after the first vaccine adminstration, the COV-P group had antibody responses higher than the COV-N participants at any timepoint ( Fig 2C) . However, in both COV-P and COV-N groups, the highest vaccineinduced antibody responses occurred one month after 2 nd dose, that is 3 months the after the first vaccination (Fig 2A) , although the COV-P group responded with 2.2 fold higher levels compared to COV-N at that time point (Fig 2D) . One month later, in COV-P vaccinees, IgG levels significantly decreased (P<0.05) but were still higher (P<0.05) than the COV-N group. Six months after initial vaccination, antibody levels further decreased in both COV-P and COV-N and no difference was observed between them at that time point. However, fold difference in the geometric mean IgG concentration (post-vaccination /pre-vaccination) was higher in the COV-N group compared to COV-P group.

Compared to pre-vaccination levels, levels 4 months after receipt of the 1 st Covishield vaccine dose, the geometric mean increase was 9.3 fold for the COV-P group while it was 23.5 for the COV-N vaccinees.

The IgG antibody responses were analyzed between males and females after Covishield vaccination. No significant difference was observed (P>0.05) between males and females at all time points after Covishield vaccination ( Fig 3A) .

Next, we analyzed vaccine induced IgG antibodies in participants in two age groups; those below 40 years of age and those 40 years and above (Fig 3B) . A comparable increase of SARS-CoV-2 specific IgG antibodies was observed in both age groups. No significant differences were observed (P>0.05) between the age groups until 5 months after the initial Covishield vaccination.

By 6 months after initial vaccination, those in the younger age group had a significantly higher level of antibody responses (P<0.05) compared to those 40 years and aboves ( Fig 3B) . However, both age groups were still 100% seropositive at 6 months after initial vaccination. Furthermore, this difference is based on a relatively smaller sample size, as compared with the earlier timepoints.

We also compared age specific vaccine responses in the COV-P and COV-N participants (Fig   4) . We observed that within the COV-N group, those participants <40 years of age had significantly higher IgG antibodies compared to those in the ≥ 40 years age group at one month (P<0.05) and two months (P<0.001) following the first vaccine dose. However, after the second dose, responses were comparable in both age groups within 3-5 months after vaccination. Again, 6 months following initial vaccination, COV-N participants in the younger age group (<40 years) had higher antibody levels compared to the older group, which confirms the importance of receiving a booster dose in the older age group 6 months following initial vaccination.

In contrast, within COV-P participants, vaccine responses were comparable in both age groups after the first dose. However, one month after the second dose, significantly higher (P<0.05) antibody responses were observed in the ≥ 40 years age group compared to those below 40 years age (Fig 4) .

We were interested in understanding if there were any differences in Covishield vaccine induced antibody responses between participants who had comorbid diseases compared to participants who did not. Covishield vaccine induced SARS-CoV-2 antibodies regardless of comorbidity status; compared to the prevaccination level, a significant (P<0.0001) rise of IgG antibodies was observed in both groups at all time points after vaccination. Participants with comorbidities had slightly higher baseline IgG seropositivity compared to participants without comorbidities (58% vs. 49%, data not shown) although rate of COVID positivity prior to vaccination was comparable in participants with (53%) and without comorbidities (57%). However, no significant differences in SARS-CoV-2 IgG levels were observed between vaccinees with comorbidity and those without at any subsequent time points (P>0.05).

In this study, we found that two doses of the Covishield vaccine given at an 8 week interval generates significant SARS-CoV-2 specific antibody responses in adults in Bangladesh. Persons who reported recent SARS-CoV-2 infection had higher levels of antibody to SARS-CoV-2 RBD following vaccination than did those without a history of infection. However, the gap between previously infected and not previously infected individuals was highest after the first dose of vaccination. Ultimately by 6 months after the iniation of vaccination, after receiving a second dose of the vaccine, there was no significant difference in titer between those with and without evidence of prior SARS-CoV-2 infection.

Without an established immune correlate of protection for SARS-CoV-2 vaccines, as well as the continued emergence of new SARS-CoV-2 variants, the protective efficacy of vaccination cannot be precisely inferred from these results. However, our antibody findings are consistent with other studies which suggest that repeated exposures to both vaccination and natural infection (eg hybrid immunity) serve to increase protection associated with vaccination (Hammerman et al., 2022) . However, our study suggests the immunologic benefits of hybrid immunity may be mitigated over time and by repeated doses of vaccines. In other words, the immunologic benefits of mixed exposure to vaccination and natural infection may be reduced over time if sufficient boosting by vaccination occurs.

Recently, the British drug manufacturer reported that a third booster shot of the Vaxzevria vaccine (same as Covishield produced by the Serum Institute of India) significantly boosts antibody levels against the Omicron variant of the coronavirus. Some experts are also of the opinion that mix and matching of vaccine shots may give better results to boost immunity (Costa Clemens et al., 2022 , Nguyen et al., 2022 , Shaw Robert H. et al., 2021 . However, the AstraZeneca has advocated the use of Vaxzevria as a third booster dose against Omicron (Gloucester Advocate, 2022) . If the vaccine is recommended for use as a booster against Omicron, it may significantly boost the demand for Covishield in India.

Different comorbid conditions (eg: hypertension, cardiac disease, kidney disease, diabetes mellitus etc) were also analyzed for observing association with differing responses following COVID-19 vaccination. However, comorbidity did not appear to have any negative effect for mounting immune responses after vaccination with Covishield. In addition, there was no significant difference in antibody responses after second dose of Covishield vaccination between participants who had comorbid condition and who did not. However, another study showed that the antibody response among comorbid individuals had lower antibody levels compared to those without comorbidity after the second dose of Vaxzevria, although the difference in the level of antibodies is very small between participants having comorbidities (median 10.60; IQR: 8.21-11.90) and no comorbidities (median 10.60; IQR: 8.21-11.90) (Hoque et al., 2021) .

Our preliminary results re-emphasize the urgent need to deploy the most effective vaccine strategies as widely and rapidly as possible in order to provide population protection against the different emerging lineages of concern of SARS-CoV-2. The Government of Bangladesh is currently trying to cover the population by mass vaccination campaigns in different divisions, cities and wards over the country. Policy makers are continuously observing outcomes from different countries and implementing improvements in our vaccine administration strategies. Therefore, our findings will help policy makers understand immune response to Covishield over time and in different age groups, by gender and by co-morbidities. Several ongoing studies are investigating the effects of mixing different COVID-19 vaccine formulations including for boosters. Data has been released from mixed trials in Asian and European countries, which suggest that mixing vaccines leads to a stronger immune response and sometimes out performs two doses of the same vaccine (Callaway, 2021 , Maccioni, 2021 , Melimopoulos, 2021 , Shaw R. H. et al., 2021 . We also observed waning of antibody responses in a few participants after 

Authors are extremely thankful to all field and laboratory staffs who accomplished a wonderful job during the pandemic by collection of blood from the community and processed the blood in the laboratory.

Informed written consent was obtained from all the participants. The Institutional Review Committee of the icddr,b and IEDCR approved the study protocol.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet and again 1 month (day 30±10, n=126), 2 months (day 60±10, n=75), 3 months (day 90±10, n=161), 4 months (day 120±10, n=32), 5 months (day 150±10, n=57) and 6 months (day 180±10, n=47) after the first dose. A second dose of vaccine was given two months after the first vaccination. Bars indicate geometric means concentration of IgG with 95% confidence interval (CI). Dashed line indicates the cut-off concentration of IgG antibody (500 ng/ml) for seropositivity. The number and percentage of seropositive individuals at each time point analyzed are shown below the graph. * P < 0.05, ** P < 0.01, *** P < 0.001, ns; not significant. and 4. ≥ 40 years and prior COVID positive (n=7-28) before vaccination and every month out until 6 months after first vaccination. A second dose of vaccine was given two months after the first vaccination. Bars indicate the geometric mean concentration of IgG with 95% confidence interval (CI). Dashed line indicates the cut-off concentration of IgG antibodies (500 ng/ml) for seropositivity. * P < 0.05, ** P < 0.01. 

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A second dose of vaccine was given two months after the first vaccination. Fold differences in the geometric mean antibody concentration between COV-N and COV-P in (B) prevaccination (C) 2 months after first vaccine dose (before second dose) and (D) 1 month after second dose (3 months after the first dose) are shown using the dashed lines. Bars indicate the geometric mean concentration of IgG with 95% confidence interval (CI). The number and percentage of seropositive individuals in each time point analyzed are shown below the graph

144 (38%) a Co-morbid diseases include asthma, hypertension, diabetes, heart diseases, kidney diseases etc. b Participants were not interested to provide co-morbidities data.