The magnitude and cross reactivity of SARS-CoV-2 specific antibody responses in vaccinated and unvaccinated Sri Lankan children and association with nutrition status (preprint)

BackgroundWe investigated the seropositivity rates of Sri Lankan children in a large island wide serosurvey, to determine the magnitude and breadth of antibody responses to SARS-CoV-2 variants and the association with the vaccination and nutritional status to understand the likely impact of newer variants in Sri Lanka. MethodsUsing the WHO UNITY protocol, we recruited 5207 children, aged 10 to 20 years, representative of the 9 provinces of Sri Lanka, and assessed seropositive rates, ACE2 blocking antibodies and antibodies to BA.2.75 and XBB.1.5, in vaccinated and unvaccinated children. Anthropometric measurements were taken to determine the association between nutrition status and antibody levels. Results3111/3119 (99.7%) vaccinated and 2008/2088 (96.2%) of unvaccinated children, were seropositive for SARS-CoV-2. 2984/3111 (95.9%) of vaccinated children had ACE2 blocking antibodies above the cut-off threshold, compared to 1346/2008 (67.0%) of unvaccinated children. 27.2 % unvaccinated children had positive antibody responses to BA.2.75 and 30.7% to XBB.1.5, while 64.3% of vaccinated had a positive response to BA.2.75 and 43.1% to XBB.1.5. Vaccinated children had significantly higher titres of total antibodies, ACE2 blocking antibodies and antibodies to XBB.1.5 and BA.2.75 than unvaccinated children. However, the vaccinated had significantly higher responses to BA.2.75 (p<0.0001), than XBB.1.5. Unvaccinated children, with <3rd BMI centile had significantly lower ACE2 blocking antibodies than other groups. DiscussionThe high seropositivity rates and antibody titres to SARS-CoV-2 variants in unvaccinated children, suggests that they are likely to have been infected more than once with SARS-CoV-2. The implications of lower antibody levels in undernourished children should be further investigated.

Identification of differences in the magnitude and specificity of SARS-CoV-2 nucleocapsid antibody responses in naturally infected and vaccinated individuals (preprint)

Background: As there are limited data on B cell epitopes for the nucleocapsid protein in SARS-CoV-2, we sought to identify the immunodominant regions within the N protein, recognized by patients with varying severity of natural infection with the Wuhan strain (WT), delta, omicron and in those who received the Sinopharm vaccines, which is an inactivated, whole virus vaccine. Methods: Using overlapping peptides representing the N protein, with an in-house ELISA, we mapped the immunodominant regions within the N protein, in seronegative (n=30), WT infected (n=30), delta infected (n=30), omicron infected+vaccinated (n=20) and Sinopharm (BBIBP-CorV) vaccinees (n=30). We then investigated the sensitivity and specificity of these immunodominant regions and analysed their conservation with other SARS-CoV-2 variants of concern, seasonal human coronaviruses and bat Sarbecoviruses. We then investigated the kinetics of responses to these regions in those with varying severity of acute COVID-19. Results: We identified four immunodominant regions aa 29-52, aa 155-178, aa 274 to 297 and aa 365 to 388, were highly conserved within SARS-CoV-2 and the bat coronaviruses. The magnitude of responses to these regions varied based on the infecting SARS-CoV-2 variants, with WT infected individuals predominantly recognizing aa155 to 178 regions, delta infected individuals and vaccinated+omicron infected individuals predominantly recognizing regions aa 29 to 52 and aa 274 to 294 regions. Sinopharm vaccinees recognized all four regions, with the magnitude of responses significantly lower than other groups. >80% of individuals gave responses above the positive cut-off threshold to many of the four regions, with some differences with individuals who were infected with different VoCs. These regions were found to be 100% specific, as none of the seronegative individuals gave any responses. Conclusions: N-protein specific responses appear to be detectable in over 90% of those who were naturally infected or vaccinated with a whole virus inactivated vaccine, with responses mainly directed against four regions of the protein, which were highly conserved. As these regions were highly specific with high sensitivity, they have a potential to be used to develop diagnostic assays and to be used in development of vaccines.

Comparison of the Immunogenicity of five COVID-19 vaccines in Sri Lanka (preprint)

We assessed antibody responses 3 months post-vaccination in those who received mRNA-1273 (n=225), Sputnik V (n=128) or the first dose of Gam-COVID-Vac (n=184) and compared the results with previously reported data of Sinopharm and AZD1222 vaccinees. 99.5% of Moderna >94% of AZD1222 or Sputnik V, 72% to 76% of Gam-COVID-Vac (first dose) and 38.1% to 68.3% of Sinopharm vaccinees had ACE2 blocking antibodies above the positive threshold. The ACE2 blocking antibody levels were highest to lowest was Moderna > Sputnik V/ AZD1222 (had equal levels)> first dose of Gam-COVID-Vac > Sinopharm. All Moderna recipients had antibodies above the positive threshold to the ancestral (WT), B.1.1.7, B.1.351.1 and 80% positivity rate for B.1.617.2. Positivity rates of Sputnik V vaccinees for WT and variants, were higher than AZD1222 vaccinees, while Sinopharm vaccinees had the lowest positivity rates (<16.7%). These findings highlight the need for further studies to understand the effects on clinical outcomes.

Kinetics of immune responses to the AZD1222/Covishield vaccine with varying dose intervals in Sri Lankan individuals (preprint)

Background To understand the kinetics of immune responses with different dosing gaps of the AZD1222 vaccine, we compared antibody and T cell responses in two cohorts with two different dosing gaps. Methods Antibodies to the SARS-CoV-2 virus were assessed in 297 individuals with a dosing gap of 12 weeks, sampled at 12 weeks post second dose (cohort 1) and in 77 individuals with a median dosing gap of 21.4 weeks (cohort 2) sampled 6 weeks post second dose. ACE2 receptor blocking antibodies (ACE2R-Abs), antibodies to the receptor binding domain (RBD) of the virus and variants of concern (VOC) and ex vivo T cell responses were assessed in a sub cohort. Results All individuals (100%) had SARS-CoV-2 specific total antibodies and 94.2% of cohort 1 and 97.1% of cohort 2 had ACE2R-blocking Abs. There was no difference in antibody titres or positivity rates in different age groups in both cohorts. The ACE2R-blocking Abs (p<0.0001) and antibodies to the RBD of the VOCs were significantly higher in cohort 2, compared to cohort 1. 41.2% to 65.8% of different age groups gave a positive response by the haemagglutination assay to the RBD of the ancestral virus and VOCs in cohort 1, while 53.6% to 90% gave a positive response in cohort 2. 17/57 (29.8%) of cohort 1 and 17/29 (58.6%) of cohort 2 had ex vivo IFNγ ELISpot responses above the positive threshold. The ACE2R-blocking antibodies and ex vivo IFNγ ELISpot responses at 12 weeks post-first dose, significantly correlated with levels 12 weeks post second dose (Spearman’s r=0.46, p=0.008) and (Spearman’s r=0.71, p<0.0001) respectively. Conclusions Both dosing schedules resulted in high levels of antibody and T cell responses post vaccination, although those with a longer dosing gap had a higher magnitude of responses, possibly as immune responses were measured 6 weeks post second dose compared to 12 weeks post second dose.

Immune responses following the first dose of the Sputnik V (Gam-COVID-Vac) (preprint)

As the first dose of Gam-COVID-Vac, is currently used as a single dose vaccine in some countries, we investigated the immunogenicity of this at 4 weeks (327 naïve individuals). 88.7% seroconverted, with significantly lower seroconversion rates in those over 60 years (p = 0.004) and significantly lower than previously seen with AZD1222 (p = 0.018). 82.6% developed ACE2 receptor blocking antibodies, although levels were significantly lower than following natural infection (p = 0.0009) and a single dose of AZD1222 (p 

Antibody and T cell responses to Sinopharm/BBIBP-CorV in naïve and previously infected individuals in Sri Lanka (preprint)

Background As there are limited data of the immunogenicity of the Sinopharm/BBIBP-CorV in different populations, antibody responses against different SARS-CoV-2 variants of concern and T cell responses, we investigated the immunogenicity of the vaccine, in individuals in Sri Lanka. Methods SARS-CoV-2-specific antibodies were measured in 282 individuals who were seronegative at baseline, and ACE2 receptor blocking antibodies, antibodies to the receptor binding domain (RBD) of the wild type (WT), B.1.1.7, B.1.351 and B.1.617.2, ex vivo and cultured IFNγ ELISpot assays, intracellular cytokine secretion assays and B cell ELISpot assays were carried out in a sub cohort of the vaccinees at 4 weeks and at 6 weeks (2 weeks after the second dose). Results 95% of the vaccinees seroconverted, although the seroconversion rates were significantly lower (p<0.001) in individuals >60 years (93.3%) compared to those who were 20 to 39 years (98.9%). 81.25% had ACE2 receptor blocking antibodies at 6 weeks, and there was no difference in these antibody titres in vaccine sera compared to convalescent sera (p=0.44). Vaccinees had significantly less (p<0.0001) antibodies to the RBD of WT and B.1.1.7, although there was no difference in antibodies to the RBD of B.1.351 and B.1.617.2 compared to convalescent sera. 27.7% of 46.4% of vaccinees had ex vivo IFNγ and cultured ELISpot responses respectively, and IFNγ and CD107a responses were detected by flow cytometry. Conclusions Sinopharm/BBIBP-CorV appeared to induce high seroconversion rates and induce a similar level of antibody responses against ACE2 receptor, B.1.617.2 and B.1.351 as seen following natural infection.

Comparison of kinetics of immune responses to SARS-CoV-2 proteins in individuals with varying severity of infection and following a single dose of the AZD1222 (preprint)

Background While there have been many studies characterizing the IgG and IgA responses to different SARS-CoV-2 proteins in individuals with natural infection, the induction of IgG and IgA to different viral proteins in vaccinees have not been extensively studied. Therefore, we sought to investigate the antibody responses to SARS-CoV-2 following natural infection and following a single dose of AZD2221, in Sri Lankan individuals. Methods Using Luminex assays, we characterized the IgG and IgA responses in patients with varying severity of illness and following a single dose of the vaccine at 4 weeks and 12 weeks since onset of illness or following vaccination. Haemagglutination test (HAT) was used to assess the antibodies to the receptor binding domain of SARS-CoV-2 wild type (WT), B.1.1.7, B.1.351 and B.1.617.2 (VOCs) and surrogate neutralizing test to measure ACE2 receptor blocking antibodies. Results Those with mild illness and in vaccinees, the IgG responses to S1, S2, RBD and N protein increased from 4 weeks to 12 weeks, while it remained unchanged in those with moderate/severe illness. Those who had a febrile illness in 2017 and 2018 (controls) also gave IgG and IgA high responses to the S2 subunit. In the vaccinees, the most significant rise was seen for the IgG antibodies to the S2 subunit (p<0.0001). Vaccinees had several fold lower IgA antibodies to all the SARS-CoV-2 proteins tested than those with mild and moderate/severe illness at 4 weeks and 12 weeks. At 12 weeks the HAT titres were significantly lower to the B.1.1.7 in vaccinees and significantly lower in those with mild illness, and in vaccinees to B.1.351 and for B.1.617.2. No such difference was seen in those with moderate/severe illness. Conclusions Vaccinees had significantly less IgA to SARS-CoV-2, but comparable IgG responses to those with natural infection. However, following a single dose, vaccinees had reduced antibody levels to the variants of concern (VOC), which further declined with time, compared to natural infection.

Seroprevalence of SARS-CoV-2 infection in the Colombo Municipality region, Sri Lanka (preprint)

Background As the Municipality Council area in Colombo (CMC) experienced the highest number of cases until end of January 2021, in Sri Lanka, we carried out a serosurvey prior to initiation of the vaccination program to understand the extent of the SARS-CoV-2 outbreak. Methods SARS-CoV-2 seropositivity was determined in 2547 individuals between the ages of 10 to 86 years, by the Wantai total antibody ELISA. We also compared to seroprevalence using the haemagglutination test (HAT) to evaluate its usefulness in carrying out serosurveys. Results The overall seropositivity rate was 24.46%, while seropositivity by HAT was 18.9%. Although the SARS-CoV-2 infection detection rates by PCR were highest in the population between the ages of 20 to 60 years of age, the seropositivity rates were equal among all age groups. The seropositivity rate was highest in the 10 to 20 age group (34.03%), whereas the PCR positivity rates was 9.8%. Differences in the PCR positivity rates and seropositivity rates were also seen in 60- to 70-year-olds (8.9% vs 30.4%) and in individuals >70 year (4.1% vs 1.2%). The seropositivity rates of the females was 29.7% (290/976), which was significantly higher (p<0.002) than in males 21.2% (333/1571). Conclusions A high seroprevalence rate (24.5%) was seen in all age groups in the CMC suggesting that a high level of transmission was seen during this area. The PCR positivity rates, appear to underestimate the true extent of the outbreak and the age groups which were infected.

Sensitivity and Specificity of Two WHO Approved SARS-CoV2 Antigen Assays in Detecting Patients with SARS-CoV2 Infection (preprint)

Background: SARS-CoV-2 rapid antigen (Ag) detection kits are widely used in addition to quantitative real-time PCR (RT-qPCR), as they are cheaper with a rapid turnaround time. As there are many concerns regarding their sensitivity and specificity, in different settings, we evaluated two WHO approved rapid Ag kits in a large cohort of Sri Lankan individuals. Methods: Paired nasopharangeal swabs were obtained from 4845 participants for validation of the SD-Biosensor rapid Ag assay and 3625 for the Abbott rapid Ag assay, in comparison to RT-qPCR. A short questionnaire was used to record symptoms at the time of testing, and blood samples were obtained from 2721 of them for detection of SARS-CoV-2 specific antibodies. Results: The overall sensitivity of the SD-Biosensor Ag kit was 36.5% and the Abbott Ag test was 50.76%. The Abbott Ag test showed specificity of 99.4% and the SD-Biosensor Ag test 97.5%. At Ct values <25, the sensitivity was 71.3% to 76.6% for the SD-Biosensor Ag test and 77.3% to 88.9% for the Abbott Ag test. The Ct values for all genes (RdRP, S, E and N) tested with all RT-qPCR kits were significantly lower for the positive results of the Abbott Ag test compared to the SD-Biosensor test. 209 (48.04%) individuals who had antibodies gave a positive RT-qPCR result, and antibody positivity rates were higher at Ct values >30 (46.1 to 82.9%). 32.1% of those who gave a positive result with the SD-Biosensor Ag test and 26.3% of those who gave positive results with the Abbott Ag test had SARS-CoV-2 antibodies at the time of detection. Conclusions: Both rapid Ag tests appeared to be highly sensitive in detecting individuals at lower Ct values, in a community setting in Sri Lanka, but it will be important to further establish the relationship to infectivity.