摘要
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.
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Sprains and Strains , Infections , COVID-19 , Hepatitis D摘要
Background: With the onset of the COVID-19 pandemic in early 2020 there was a drastic reduction in the number of dengue cases in Sri Lanka, with an increase towards the end of 2021. We sought to study the contribution of virological factors, human mobility, school closure and mosquito factors in affecting these changes in dengue transmission in Sri Lanka during this time. Methods and findings: To understand the reasons for the differences in the dengue case numbers in 2020 to 2021 compared to previous years, we determined the association between the case numbers in Colombo (which has continuously reported the highest number of cases) with school closures, stringency index, changes in dengue virus (DENV) serotypes and vector densities. There was a 79.4% drop in dengue cases from 2019 to 2020 in Colombo. A significant negative correlation was seen with the number of cases and school closures (Spearmans r=-0.4732, p=<0.0001) and a negative correlation, which was not significant, between the stringency index and case numbers (Spearmans r= -0.3755 p=0.0587). There was no change in the circulating DENV serotypes with DENV2 remaining the most prevalent serotype by early 2022 (65%), similar to the frequencies observed by end of 2019. The Aedes aegypti premise and container indices showed positive but insignificant correlations with dengue case numbers (Spearman r= 0.8827, p=0.93). Conclusions: Lockdown measures, especially school closures seemed to have had a significant impact on the number of dengue cases, while the vector indices had a limited effect.
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COVID-19摘要
Background: The worst SARS-CoV-2 outbreak in Sri Lanka was due to the two Sri Lankan delta sub-lineages AY.28 and AY.104. We proceeded to further characterize the mutations and clinical disease severity of these two sub-lineages. Methods: 705 delta SARS-CoV-2 genomes sequenced by our laboratory from mid-May to November 2021 using Illumina and Oxford Nanopore were included in the analysis. The clinical disease severity of 440/705 individuals were further analyzed to determine if infection with either AY.28 or AY.104 was associated with more severe disease. Sub-genomic RNA (sg-RNA) expression was analyzed using periscope. Results: AY.28 was the dominant variant throughout the outbreak, accounting for 67.7% of infections during the peak of the outbreak. AY.28 had three lineage defining mutations in the spike protein: A222V (92.80%), A701S (88.06%), and A1078S (92.04%) and seven in the ORF1a: R24C, K634N, P1640L, A2994V, A3209V, V3718A, and T3750I. AY.104 was characterized by the high prevalence of T95I (90.81%) and T572L (65.01%) mutations in the spike protein and by the absence of P1640L (94.28%) in ORF1a with the presence of A1918V (98.58%) mutation. The mean sgRNA expression levels of ORF6 in AY.28 were significantly higher compared to AY.104 (p < 0.0001) and B.1.617.2 (p < 0.01). Also, ORF3a showed significantly higher sgRNA expression in AY.28 compared to AY.104 (p < 0.0001). There was no difference in the clinical disease severity or duration of hospitalization in individuals infected with these sub lineages. Conclusions: Therefore, AY.28 appears to have a fitness advantage over the parental delta variant (B.1.617.2) and AY.104 possibly due to the A222V mutation. AY.28 also had a higher expression of sg-RNA compared to other sub-lineages. The clinical implications of these should be further investigated.
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Seizures摘要
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.
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COVID-19摘要
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.
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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
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Introduction: Due to limited access to vaccines, many countries have only administered a single dose of the AZD1222, while the dosage intervals have increased [≥] weeks. We sought to investigate the immunogenicity of a single dose of vaccine at [≥] 16 weeks. Methods: SARS-CoV-2 specific antibodies in 553 individuals and antibodies to the receptor binding domain (RBD) of the Wuhan virus (WT) and the variants of concern (VOCs), ACE2 receptor blocking antibodies, ex vivo and cultured IFN{gamma} T cell responses and B cell ELISpot responses were investigated in a sub-cohort. Results: The seropositivity rates in those >70 years of age (93.7%) was not significantly different compared to other age groups (97.7 to 98.2, Pearson Chi-Square = 7.8, p-value = 0.05). The antibody titres (antibody index) significantly declined (p<0.0001) with increase in age. 18/69 (26.1%) of individuals did not have ACE2 receptor blocking antibodies, while responses to the RBD of WT (p=0.03), B.1.1.7 (p=0.04) and B.1.617.2 (p=0.02) were significantly lower in those who were >60 years. Ex vivo IFN {gamma} T cell ELISpot responses were seen in 10/66 (15.1%), while only a few expressed CD107a. However, >85% had a high frequency of cultured IFN{gamma} T cell ELISpot responses and B cell ELISpots. Conclusion: Virus specific antibodies were maintained at [≥] 16 weeks after receiving a single dose of AZD1222, although levels were lower to VOCs, especially in older individuals. A single dose induced a high frequency of memory T and B cell responses.
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Memory Disorders摘要
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.
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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.
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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.
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COVID-19摘要
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.
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BackgroundIn order to determine the immunogenicity of a single dose of the AZD1222/Covishield vaccine in a real-world situation, we assessed the immunogenicity, in a large cohort of health care workers in Sri Lanka. MethodsSARS-CoV-2 antibodies was carried out in 607 naive and 26 previously infected health care workers (HCWs) 28 to 32 days following a single dose of the vaccine. Haemagglutination test (HAT) for antibodies to the receptor binding domain (RBD) of the wild type virus, B.1.1.7, B.1.351 and the surrogate neutralization assay (sVNT) was carried out in 69 naive and 26 previously infected individuals. Spike protein (pools S1 and S2) specific T cell responses were measured by ex vivo ELISpot IFN{gamma} assays in 76 individuals. Results92.9% of previously naive HCWs seroconverted to a single dose of the vaccine, irrespective of age and gender; and ACE2 blocking antibodies were detected in 67/69 (97.1%) previously naive vaccine recipients. Although high levels of antibodies were found to the RBD of the wild type virus, the titres for B.1.1.7 and B.1.351 were lower in previously naive HCWs. Ex vivo T cell responses were observed to S1 in 63.9% HCWs and S2 in 31.9%. The ACE2 blocking titres measured by the sVNT significantly increased (p<0.0001) from a median of 54.1 to 97.9 % of inhibition, in previously infected HCWs and antibodies to the RBD for the variants B.1.1.7 and B.1.351 also significantly increased. Discussiona single dose of the AZD1222/Covishield vaccine was shown to be highly immunogenic in previously naive individuals inducing antibody levels greater than following natural infection. In infected individuals, a single dose induced very high levels of ACE2 blocking antibodies and antibodies to RBDs of SARS-CoV-2 variants of concern. FundingWe are grateful to the World Health Organization, UK Medical Research Council and the Foreign and Commonwealth Office.