The emergence of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) variants of concern (VOCs) has contributed to the increased morbidity and mortality caused by the COVID-19 (coronavirus disease 2019) pandemic [1
]. Among the growing number of VOCs, the beta (B.1.351) variant was first detected in South Africa and drove the second SARS-CoV-2 epidemic wave in southern Africa, which was associated with a substantial increase in cases and deaths compared to the first wave [2
]. The beta variant bears genetic changes in the functional domain of the SARS-CoV-2 spike (S) protein including substitutions in the receptor-binding domain (RBD) (E484K, N501Y and K417N), four substitutions and a deletion in N-terminal domain (NTD) (L18F, D80A, D215G, L242H and R246I) and substitutions in S2 (D614G and A701V) regions [2
]. Consequentially, variations in the RBD of the Spike protein, a classic feature of VOCs, not only increases affinity for the ACE2 human receptor [3
] but also aids immune evasion [5
Neutralisation activity against the beta variant was reduced by 13-fold in convalescent sera from individuals infected with the original D614G variant [6
]. Furthermore, vaccine-induced antibody neutralisation by the AstraZeneca COVID-19 vaccine was reduced against the beta variant compared to the original D614G variant [8
]. As a result, AstraZeneca COVID-19 vaccine showed poor efficacy against mild/moderate beta variant infection [8
], but efficacy against severe COVID-19 was not assessed because of the small number of outcomes, while, in a real-life setting, vaccine effectiveness against any symptomatic disease 21 days post first dose of the AstraZeneca COVID-19 vaccine was 50% against the beta/gamma (P.1) variants and 70% and 72% against the delta (B.1.617.2) and alpha (B.1.1.7) variants, respectively [9
]. However, vaccine effectiveness against hospitalisation or death was 82% against the beta/gamma variants, and 87% and 90% against the delta and alpha variants, respectively [9
]. These studies highlight the immune escape potency of the beta variant, which has now also been seen with other VOCs that have appeared since, including the delta and omicron (B.1.1.529) variants.
Multiple studies have reported that COVID-19 vaccination of individuals who were previously infected with SARS-CoV-2 induces robust cellular and antibody responses [9
], termed hybrid immunity [12
]. Receptor-binding domain (RBD)-specific memory B cells were increased 5- to 10-fold in hybrid immunity compared with natural infection or vaccination alone in two studies [10
]. Neutralising antibody titres induced following a single COVID-19 mRNA vaccine dose in previously infected individuals was 20-fold higher than after two doses of same vaccine in naïve individual. Moreover, these antibodies were shown to be cross-neutralising across multiple VOCs, including alpha and beta [10
]. Recently, it has been shown that breakthrough infections in vaccinated individuals also demonstrate a hybrid immunity enhanced phenotype [16
], observed in individuals vaccinated following prior SARS-CoV-2 infection [10
]. The increase in cross variant-neutralising antibodies induced by hybrid immunity is thought to be due to a recall response of diverse and high-quality memory B cells generated against natural infection [12
]. However, most of these studies have focused on mRNA vaccines, relied on primary infection induced by the ancestral variant, and have been done in non-African populations. To our knowledge, the only published adenovirus-based vaccine study done in an African population was done using the Johnson and Johnson Ad26.COV2.S single-dose vaccine in South Africa, which showed vaccination following prior infection significantly boosts spike-binding antibodies, antibody-dependent cellular cytotoxicity, and neutralising antibodies against D614G, beta, and delta VOCs [18
Here, we report an assessment of the dynamics of anti-SARS-CoV-2 antibodies in an African adult population with prior SARS-CoV-2 infection and subsequently receiving a single dose of AstraZeneca COVID-19 vaccine. These data offer important insights on natural and vaccine-induced antibody responses against VOCs and highlight the potency of hybrid immunity induced after vaccination with an adenovirus-based vaccine in previously mild/moderate SARS-CoV-2 infected adults.
Study setting and population
Using a prospective study design, recovered mild/moderate COVID-19 adult patients were recruited during the first two epidemic waves in Malawi (Blantyre City, southern region) and followed up every 30 days for a maximum of 270 days. We used a convenience sampling approach, whereby the study was advertised electronically and by word of mouth, with support from the Blantyre District Health Office and Malawi-Liverpool-Wellcome programme. Inclusion criteria for the study included being a Blantyre resident, aged between 18 and 65 years old, and having previous history of laboratory-confirmed diagnosis of COVID-19 not less than 28 days at the time of recruitment. The exclusion criteria included withholding consent and having symptoms suggestive of COVID-19 at time of recruitment. Peripheral blood samples were collected at recruitment and subsequent follow-ups. We used electronic case report forms (eCRFs) to collect clinical history and demographic data. The first wave in Malawi peaked in July 2020 and the second wave peaked in January 2021, driven by the original variant and Beta variant, respectively [19
Semi-quantitative SARS-CoV-2 Spike (S) and receptor-binding domain (RBD) IgG antibody enzyme-linked immunosorbent assay
The SARS-CoV-2 original (D614G) spike and RBD proteins were expressed in human embryonic kidney (HEK) 293F suspension cells by transfecting the cells with the spike plasmid. After incubating for 6 days at 37 °C, 70% humidity and 10% CO2, proteins were purified using a nickel resin followed by size-exclusion chromatography. Relevant fractions were collected and flash-frozen until use. Spike or RBD protein (2 μg/ml) was used to coat 96-well, high-binding plates and incubated overnight at 4 °C. The plates were incubated in a blocking buffer consisting of 5% skimmed milk powder, 0.05% Tween 20, 1x PBS. Plasma samples were diluted to a 1:100 starting dilution in blocking buffer and added to the plates. Secondary antibody was diluted to 1:3000 in blocking buffer and added to the plates followed by TMB substrate (Thermofisher Scientific). Upon stopping the reaction with 1 M H2SO4, absorbance was measured at a 450-nm wavelength. In all instances, the CR3022 mAb was used as a positive control and palivizumab was used as a negative control.
Luminex-based quantitation of SARS-CoV-2 full-length spike and RBD IgG antibodies
The assay was performed as previously reported [21
]. In brief, the expression plasmid encoding for SARS-CoV-2 RBD and full-length Spike were obtained from the Florian Krammer, Mount Sinai, USA. The recombinant trimeric Spike and RBD proteins were expressed as described previously [22
] and were coupled to the magnetic microsphere beads (Bio-Rad, USA) using a two-step carbodiimide reaction [23
]. An in-house references serum was developed by pooling convalescent serum from adult COVID-19 positive patients. This interim reference serum was calibrated against research reagent NIBSC 20/130 distributed by the National Institute for Standards and Biological Control (NIBSC) for the purpose of development and evaluation of serological assays for the detection of antibodies against SARS-CoV-2 (NIBSC, Potters Bar, UK). The binding antibody units (BAU) values assigned to in-house reference serum were 1242 BAU/mL and 2819 BAU/mL for RBD and full-length Spike IgG, respectively. Serum samples collected before 2020 were used for the analysis of assay specificity. Values of 26 BAU/mL and 32 BAU/mL were selected as the threshold indicative of SARS-CoV-2 antibodies, based on the highest value of RBD and full-length Spike IgG in samples from pre-COVID-19. Sensitivity of the assay in detecting past or current infection was assessed using serum samples obtained from randomly selected participants (n
= 15) who tested SARS-CoV-2 PCR positive and who had serial sampling before and after post-symptom onset, including cases with mild-moderate illness and asymptomatic infections. The sensitivity of the IgG assay was 75% for samples taken 7–14 days and 100 % for samples taken above 14 days following the PCR positive for SARS-CoV-2 for both RBD and full-length Spike IgG. The assay was also evaluated against a COVID-19 convalescent plasma panel (NIBSC code 20/118) intended for the development and evaluation of serological assays for the detection of antibodies against SARS-CoV-2. The in-house characterisation of the plasma panel was in line with recommended criteria with 20/120 having the highest anti-RBD antibody titre followed by 20/122, which had a mid-antibody titre, 20/124 and 20/126 had the lowest titres, and 20/128 as negative. The optimal serum/plasma and secondary antibody dilutions for the assay were 1:100 and 1:200, respectively. The over-range samples were re-tested at higher dilutions (1:200-1:1000). Samples were analysed in duplicate, and each plate included two in-house control sera. Bead fluorescence was read with the Bio-Plex 200 instrument (Bio-Rad) using Bio-Plex manager 5.0 software (Bio-Rad).
Haemagglutination test for detection of antibodies to SARS-CoV-2 variants of concern
The haemagglutination test (HAT) has been developed in Prof Alain Townsend’s (AT) laboratory at the University of Oxford and uses the IH4-RBD. The IH4-RBD reagent is based on the camelid nanobody VHH-IH4, linked to the RBD of SARS-2 Spike protein. IH4 is specific for a conserved epitope on Glycophorin A, comprised of residues 52–55 (YPPE). The IH4-RBD fusion was designed by AT and has been produced by Absolute Antibody (Oxford, UK) in bulk (1g). One milligram is enough for 10,000 tests (100 ng/well). The RBD proteins were derived from the original D614G strain and the four variants of concern, namely alpha, beta, gamma, and delta. We conducted the assay as previously reported [24
]. In brief, O negative red blood cells obtained from the Malawi Blood Transfusion Services were diluted in phosphate-buffered saline (PBS) at 1:20 and plated in V-bottomed 96 well plates. Doubling dilution of serum samples starting from 1:20 were added to the plate until 1:640 dilution. CR3022 (a human monoclonal antibody isolated from a SARS recovered patient) and EY-6A (an antibody isolated from a COVID-19 recovered patient) reagents were used as positive controls, as they all bind to similar epitopes on RBD of all the variants, allowing cross-linking between red blood cells (RBCs) labelled with IH4-RBD. IH4-RBD was added to each well and RBCs were allowed to settle for an hour. The plate was then tilted for at least 30 seconds and photographed. The HAT titre was defined by the last well in which the teardrop fails to form. Partial teardrop was regarded as negative.
Pseudovirus neutralisation assay
Samples that were seropositive for anti-Spike binding antibodies in the semi-quantitative ELISA were screened for neutralising activity as previously described [6
]. SARS-CoV-2-pseudotyped lentiviruses were prepared by co-transfecting the HEK 293T cell line with either the SARS-CoV-2 original spike (D614G) or the SARS-CoV-2 beta or delta spike plasmids in conjunction with a firefly luciferase encoding pNL4 lentivirus backbone plasmid. The parental plasmids were kindly provided by Drs. Elise Landais and Devin Sok (The International AIDS Vaccine Initiative (IAVI), USA). For the neutralisation assay, heat-inactivated seropositive serum samples were incubated with the SARS- CoV-2 pseudotyped virus for 1 h at 37 °C, 5% CO2
. Subsequently, 1 × 104 HEK 293 T cells that were engineered to over-express ACE-2, kindly provided by Dr. Michael Farzan (Scripps Research), were added and incubated at 37 °C, 5% CO2
for 72 h upon which the luminescence of the luciferase gene was measured.
Data visualisation and statistical analyses were performed in GraphPad Prism software (version 9.1.2). The antibody binding and neutralisation activity data were log10 transformed. Ordinary or repeated measures one-way ANOVA was used to compare log10 transformed data, with p value adjusted for multiple comparisons using Šídák’s or Holm-Šídák’s multiple comparisons test. Effects were considered statistically significant when the p-value was less than 0.05.
In this study, we report binding and neutralisation antibody responses induced by SARS-CoV-2 infection and augmented by a single dose of the AstraZeneca COVID-19 vaccine. We show that antibody pseudovirus neutralisation activity induced following SARS-CoV-2 infection wanes within 6 months post laboratory-confirmed diagnosis of mild/moderate COVID-19. High concentrations of binding anti-Spike and anti-RBD IgG antibodies are associated with the presence of pseudovirus neutralisation activity in convalescent serum. Most importantly, vaccination with AstraZeneca COVID-19 vaccine in individuals with prior SARS-CoV-2 infection induced robust binding, cross-reactive, and cross-neutralising antibody responses against multiple VOCs.
mRNA vaccination in previously infected individuals induce robust cross-reactive antibody responses against SARS-CoV-2 [9
]. Consistent with these observations, we show that an adenovirus vaccine elevates levels of anti-Spike and anti-RBD antibodies that are cross-reactive against D614G, alpha, beta, gamma, and delta variants in individuals previously infected with SARS-CoV-2. We also observed cross-neutralisation across VOCs in the vaccinated individuals. In agreement, studies done in the UK (Vaxzervria) and India (COVISHIELD) also show that a single dose of the AstraZeneca COVID-19 vaccine induces high antibody titres in individuals with previous SARS-CoV-2 exposure [13
]. Of note, the UK study showed a significant increase in neutralising antibody titres to VOCs including alpha, beta, and gamma, but with the least increase observed for the beta and gamma variants [13
]. Considering that in vitro neutralising activity is highly predictive of immune protection from symptomatic SARS-CoV-2 infection [27
], presence of cross-neutralising antibodies following vaccination with AstraZeneca COVID-19 vaccine in recovered patients could potentially confer cross-protection against multiple variants.
Anti-Spike IgG antibody concentrations between 60 BAU/ml to 154 BAU/ml following vaccination were recently suggested as a potential threshold for protective immunity against symptomatic COVID-19 [26
]. In our study, a single dose of AstraZeneca COVID-19 vaccine resulted in anti-Spike IgG antibodies concentrations of 12 times above the upper limit of the proposed protective threshold of 154 BAU/ml. Moreover, following SARS-CoV-2 infection, we show high pseudovirus neutralisation activity against the infecting variant in individuals with anti-Spike IgG antibodies of greater than 62 BAU/ml. However, levels of pseudovirus neutralising antibodies induced following mild/moderate SARS-CoV-2 infection declined significantly within 6 months post diagnosis, which agrees with published literature [29
]. Notably, in those who experienced reinfection in our cohort, the timing of the reinfection occurred at the time point when there was loss of serum pseudovirus neutralisation activity against the reinfecting variant, underscoring the importance of variant-specific pseudovirus neutralising antibodies in protection from SARS-CoV-2 infection. However, in one of the reinfected individuals, the binding anti-Spike and anti-RBD IgG antibody concentrations in the last sample before reinfection were above the threshold of 154 BAU/ml, indicating that binding antibody thresholds may not work universally across variants.
Interestingly, one of the vaccinated individuals did not reach the proposed putative protective threshold of 154 BAU/ml following a single dose of the AstraZeneca COVID-19 vaccine. This individual had the lowest anti-Spike (144 BAU/ml) and anti-RBD (142 BAU/ml) binding antibodies in convalescent serum pre-vaccination among the vaccinated participants and had the lowest breadth in the HAT assay (participant #4). This finding suggests that hybrid immunity may be influenced by the baseline antibody response at the time of vaccination and warrants further investigation.
This study has considerable strengths including the availability of both binding and neutralisation data from an underrepresented sub-Saharan African population; however, there are some important limitations. First, the analysis on longevity was cross sectional, and this could impact the accuracy of our estimates on the duration of binding and neutralisation antibodies. Moreover, we have only measured binding and neutralisation function in this study, but there are other Fc-mediated antibody functions [31
], as well as memory T and B cell responses [32
], that have previously been implicated to contribute to the control of SARS-CoV-2 infection. Nevertheless, binding and neutralising antibodies are the most well characterised potential correlates of protection (CoP) against COVID-19 to date [26
]. Second, the sample size, especially for the vaccinated individuals, is small, and this may limit the generalisability of the findings. However, the consistency of the results across multiple assay platforms and their agreement with other similar published studies in other populations attests to the robustness of the findings and highlights their relevance to the field.
We report waning of pseudovirus neutralising activity within 6 months post laboratory-confirmed diagnosis of mild/moderate COVID-19 and a robust antibody response following partial vaccination with the AstraZeneca COVID-19 vaccine in adults previously infected with SARS-CoV-2. These data could have implications for COVID-19 vaccination policy as they provide further evidence highlighting the potency of hybrid immunity induced following vaccination with AstraZeneca vaccine, which is the most common vaccine in sub-Saharan Africa. However, there are still outstanding questions that needs to be addressed, which include whether partial vaccination could also induce robust humoral responses in those with prior asymptomatic infection and how long the enhanced hybrid immunity last, as these could help inform subsequent vaccination regimens or timings.
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