Humoral and T cell responses to SARS-CoV-2 reveal insights into immunity during the early pandemic period in Pakistan.

BACKGROUND: Protection against SARS-CoV-2 is mediated by humoral and T cell responses. Pakistan faced relatively low morbidity and mortality from COVID-19 through the pandemic. To examine the role of prior immunity in the population, we studied IgG antibody response levels, virus neutralizing activity and T cell reactivity to Spike protein in a healthy control group (HG) as compared with COVID-19 cases and individuals from the pre-pandemic period (PP). METHODS: HG and COVID-19 participants were recruited between October 2020 and May 2021. Pre-pandemic sera was collected before 2018. IgG antibodies against Spike and its Receptor Binding Domain (RBD) were determined by ELISA. Virus neutralization activity was determined using a PCR-based micro-neutralization assay. T cell - IFN-γ activation was assessed by ELISpot. RESULTS: Overall, the magnitude of anti-Spike IgG antibody levels as well as seropositivity was greatest in COVID-19 cases (90%) as compared with HG (39.8%) and PP (12.2%). During the study period, Pakistan experienced three COVID-19 waves. We observed that IgG seropositivity to Spike in HG increased from 10.3 to 83.5% during the study, whilst seropositivity to RBD increased from 7.5 to 33.3%. IgG antibodies to Spike and RBD were correlated positively in all three study groups. Virus neutralizing activity was identified in sera of COVID-19, HG and PP. Spike reactive T cells were present in COVID-19, HG and PP groups. Individuals with reactive T cells included those with and without IgG antibodies to Spike. CONCLUSIONS: Antibody and T cell responses to Spike protein in individuals from the pre-pandemic period suggest prior immunity against SARS-CoV-2, most likely from cross-reactive responses. The rising seroprevalence observed in healthy individuals through the pandemic without known COVID-19 may be due to the activation of adaptive immunity from cross-reactive memory B and T cells. This may explain the more favourable COVID-19 outcomes observed in this population.

Investigating the impact of prior COVID-19 on IgG antibody and interferon γ responses after BBIBP-CorV vaccination in a disease endemic population: A prospective observational study.

Background and Aims: COVID-19 vaccinations have reduced morbidity and mortality from the disease. Antibodies against severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) have been associated with immune protection. Seroprevalence studies revealed high immunoglobulin G (IgG) antibody levels to SARS-CoV-2 in the Pakistani population before vaccinations. We investigated the effect of BBIBP-CorV vaccination on circulating IgG antibodies and interferon (IFN)-γ from T cells measured in a cohort of healthy individuals, with respect to age, gender, and history of COVID-19. Methods: The study was conducted between April and October 2021. BBIBP-CorV vaccinated participants were followed up to 24 weeks. Antibodies to SARS-CoV-2 Spike protein and its receptor-binding domain (RBD) were measured. IFNγ secreted by whole blood stimulation of Spike protein and extended genome antigens was determined. Results: Study participants with a history of prior COVID-19 displayed a higher magnitude of IgG antibodies to Spike and RBD. IgG seropositivity was greater in those with prior COVID-19, aged 50 years or younger and in females. At 24 weeks after vaccination, 37.4% of participants showed IFN-γ responses to SARS-CoV-2 antigens. T cell IFN-γ release was higher in those with prior COVID-19 and those aged 50 years or less. Highest IFN-γ release was observed to extended genome antigens in individuals both with and without prior COVID-19. Conclusion: We found that IgG seropositivity to both Spike and RBD was affected by prior COVID-19, age and gender. Importantly, seropositive responses persisted up to 24 weeks after vaccination. Persistence of vaccine induced IgG antibodies may be linked to the high seroprevalence observed earlier in unvaccinated individuals. Increased T cell reactivity to Spike and extended genome antigens reflects cellular activation induced by BBIBP-CorV. COVID-19 vaccination may have longer lasting immune responses in populations with a higher seroprevalence. These data inform on vaccination booster policies for high-risk groups.

IgG antibodies to SARS-CoV-2 in asymptomatic blood donors at two time points in Karachi.

INTRODUCTION: An estimated 1.5 million cases were reported in Pakistan until 23 March, 2022. However, SARS-CoV-2 PCR testing capacity has been limited and the incidence of COVID-19 infections is unknown. Volunteer healthy blood donors can be a control population for assessment of SARS-CoV-2 exposure in the population. We determined COVID-19 seroprevalence during the second pandemic wave in Karachi in donors without known infections or symptoms in 4 weeks prior to enrollment. MATERIALS AND METHODS: We enrolled 558 healthy blood donors at the Aga Khan University Hospital between December 2020 and February 2021. ABO blood groups were determined. Serum IgG reactivity were measured to spike and receptor binding domain (RBD) proteins. RESULTS: Study subjects were predominantly males (99.1%) with a mean age of 29.0±7.4 years. Blood groups were represented by; B (35.8%), O (33.3%), A (23.8%) and AB (7%). Positive IgG responses to spike were detected in 53.4% (95% CI, 49.3-37.5) of blood donors. Positive IgG antibodies to RBD were present in 16.7% (95% CI; 13.6-19.8) of individuals. No significant difference was found between the frequency of IgG antibodies to spike or RBD across age groups. Frequencies of IgG to Spike and RBD antibodies between December 2020 and February 2021 were found to be similar. Seropositivity to either antigen between individuals of different blood groups did not differ. Notably, 31.2% of individuals with IgG antibodies to spike also had IgG antibodies to RBD. Amongst donors who had previously confirmed COVID-19 and were seropositive to spike, 40% had IgG to RBD. CONCLUSIONS: Our study provides insights into the seroprevalence of antibodies to COVID-19 in a healthy cohort in Karachi. The differential dynamics of IgG to spike and RBD likely represent both exposure to SARS-CoV-2 and associate with protective immunity in the population.

Synthesis of Isatin Derivatives Exhibiting Antibacterial, Antifungal and Cytotoxic Activities.

OBJECTIVE: The current objective was to synthesize biologically active Isatin derivatives. For this purpose, six (06) chemical entities of Isatin derivatives such as 3a-3c and 4a-4c were synthesized from Isatin substrate. METHODS: The pure compounds were characterized with the help of 1H-NMR, FT-IR, EIMS spectroscopic techniques. The synthesized amines 3a-3c and hydrazones 4a-4c were evaluated for their toxicity potential with the help of brine shrimp bioassay. RESULTS: The obtained results revealed that 3c exhibited promising toxicity (LD50 = 1.03 × 10-5 M) against Artemia salina. The pure chemical entities such as compounds 3a-3c and 4a-4c were evaluated against four Gram-negative (E. coli, P. aeruginosa, S. typhi, S. flexneri) and two Gram-positive (B. subtilis, S. aureus) bacteriae. The compound 3a displayed significant activity against two bacterial strains; i.e. P. aeruginosa, S. typhi, compound 3c demonstrated significant activity against P. aeruginosa. Compound 4b was the most active against S. typhi, displaying a greater diameter of the growth of inhibition zone (GIZ) than the standard drug. Compound 4c exhibited significant activity against P. aeruginosa and S. typhi. Compounds 3a-3c and 4a-4c exhibited moderate to significant antibacterial activity against three out of five strains. Only Compound 3c revealed promising toxicity (LD50 = 1.03 × 10-5 M) against Artemia salina. Antifungal studies of the compounds 3a-3c and 4a-4c against six fungal cultures; T. longifusus, C. albicans, A. flavus, M. canis, F. solani, and C. glabrata. All the compounds were active against one or more pathogens having inhibition (10- 90 %). Compound 3a exhibited significant inhibition i.e. 90 % against M. canis, 70 % against T. longifusus, compound 4b further displayed significant inhibition (90 %) against A. flavus and thus proved to be the most active member of the series. CONCLUSION: All compounds showed better antibacterial, antifungal and cytotoxic activities, which may provide a ground to prepare enhanced molecules with much higher antibacterial activities.

A rapid real-time polymerase chain reaction-based live virus microneutralization assay for detection of neutralizing antibodies against SARS-CoV-2 in blood/serum.

BACKGROUND: Individuals recovering from COVID-19 are known to have antibodies against the Spike and other structural proteins. Antibodies against Spike have been shown to display viral neutralization. However, not all antibodies against Spike have neutralizing ability although they may be cross-reactive. There is a need for easy-to-use SARS-CoV-2 neutralizing assays for the determination of virus-neutralizing activity in sera of individuals. Here we describe a PCR-based micro-neutralization assay that can be used to evaluate the viral neutralization titers of serum from SARS-CoV-2 infected individuals. METHODS: The SARS-CoV-2 strain used was isolated from a nasopharyngeal specimen of a COVID-19 case. The limiting dilution method was used to obtain a 50% tissue culture infective dose (TCID50) of Vero cells. For the micro-neutralization assay, 19 serum samples, with positive IgG titers against Spike Receptor-Binding Domain (RBD) were tested. After 24 hours, infected cells were inspected for the presence of a cytopathic effect, lysed and RNA RT-PCR conducted for SARS-CoV-2. PCR target Ct values were used to calculate percent neutralization/inhibition of SARS-CoV-2. RESULTS: Out of 19 samples, 13 samples gave 100% neutralization at all dilutions, 1 sample showed neutralization at the first dilution, 4 samples showed neutralization at lower dilutions, while one sample did not demonstrate any neutralization. The RBD ODs and neutralization potential percentages were found to be positively correlated. CONCLUSION: We describe a rapid RT-PCR-based SARS-CoV-2 microneutralization assay for the detection of neutralizing antibodies. This can effectively be used to test the antiviral activity of serum antibodies for the investigation of both disease-driven and vaccine-induced responses.