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.

Tracking SARS-CoV-2 variants through pandemic waves using RT-PCR testing in low-resource settings.

COVID-19 resulted in extensive morbidity and mortality worldwide. SARS-CoV-2 evolved rapidly, with increasing transmission due to Variants of Concern (VOC). Identifying VOC became important but genome submissions from low-middle income countries (LMIC) remained low leading to gaps in genomic epidemiology. We demonstrate the use of a specific mutation RT-PCR based approach to identify VOC in SARS-CoV-2 positive samples through the pandemic in Pakistan. We selected 2150 SARS-CoV-2 PCR positive respiratory specimens tested between April 2021 and February 2022, at the Aga Khan University Hospital Clinical Laboratories, Karachi, Pakistan. Commercially available RT-PCR assays were used as required for mutations in Spike protein (N501Y, A570D, E484K, K417N, L452R, P681R and deletion69_70) to identify Alpha, Beta, Gamma, Delta, and Omicron variants respectively. Three pandemic waves associated with Alpha, Delta and Omicron occurred during the study period. Of the samples screened, VOC were identified in 81.7% of cases comprising mainly; Delta (37.2%), Alpha (29.8%) and Omicron (17.1%) variants. During 2021, Alpha variants were predominant in April and May; Beta and Gamma variants emerged in May and peaked in June; the Delta variant peaked in July and remained predominant until November. Omicron (BA.1) emerged in December 2021 and remained predominant until February 2022. The CT values of Alpha, Beta, Gamma and Delta were all significantly higher than that of Omicron variants (p<0.0001). We observed VOC through the pandemic waves using spike mutation specific RT-PCR assays. We show the spike mutation specific RT-PCR assay is a rapid, low-cost and adaptable for the identification of VOC as an adjunct approach to NGS to effectively inform the public health response. Further, by associating the VOC with CT values of its diagnostic PCR we gain information regarding the viral load of samples and therefore the level of transmission and disease severity in the population.

Diagnostic accuracy of different cut-off values of adenosine deaminase levels in tuberculous pleural effusion.

Objectives: To assess the diagnostic accuracy of different cut-off values of pleural fluid adenosine deaminase levels as a diagnostic method for tuberculous pleural effusion. METHODS: The prospective study was conducted from 2014 to 2016 at the Aga Khan University Hospital, Karachi, and comprised pleural fluid samples of adult patients with and without tuberculosis which were tested for adenosine deaminase levels, and divided into tuberculosis group A and non-tuberculosis group B. Sensitivity, specificity, negative predictive value and positive predictive value were calculated using different cut-offs. Data was analysed using IBM SPSS (Statistical Package for Social Sciences) version 21.0 (IBM Corp., Armonk, NY). RESULTS: Of 155 patients, 46(29.7%) had tuberculosis; 30(65.2%) males and 16(34.8%) females. Those who did not have tuberculosis were 109(70.3%); 69(63.3%) males and 40(36.7%) females. The adenosine deaminase levels were elevated in group A compared to group B (p<0.001). The cut-off of 30U/L showed the highest sensitivity (71.7%) and negative predictive value (87.4%), and a specificity of 82.6%. The cut-off of 50U/L showed the highest specificity (89.9%) with sensitivity 52.2%, and the cut-off of 40U/L showed the highest positive predictive value of 68.9% with sensitivity 67.4% and specificity 87.2%. CONCLUSIONS: Pleural fluid adenosine deaminase testing for diagnosing tuberculosis pleuritis revealed highest sensitivity and moderate specificity for cut-off value of 30U/L.

Immune mapping of human tuberculosis and sarcoidosis lung granulomas.

Tuberculosis (TB) and sarcoidosis are both granulomatous diseases. Here, we compared the immunological microenvironments of granulomas from TB and sarcoidosis patients using in situ sequencing (ISS) transcriptomic analysis and multiplexed immunolabeling of tissue sections. TB lesions consisted of large necrotic and cellular granulomas, whereas "multifocal" granulomas with macrophages or epitheloid cell core and a T-cell rim were observed in sarcoidosis samples. The necrotic core in TB lesions was surrounded by macrophages and encircled by a dense T-cell layer. Within the T-cell layer, compact B-cell aggregates were observed in most TB samples. These B-cell clusters were vascularized and could contain defined B-/T-cell and macrophage-rich areas. The ISS of 40-60 immune transcripts revealed the enriched expression of transcripts involved in homing or migration to lymph nodes, which formed networks at single-cell distances in lymphoid areas of the TB lesions. Instead, myeloid-annotated regions were enriched in CD68, CD14, ITGAM, ITGAX, and CD4 mRNA. CXCL8 and IL1B mRNA were observed in granulocytic areas in which M. tuberculosis was also detected. In line with ISS data indicating tertiary lymphoid structures, immune labeling of TB sections expressed markers of high endothelial venules, follicular dendritic cells, follicular helper T cells, and lymph-node homing receptors on T cells. Neither ISS nor immunolabeling showed evidence of tertiary lymphoid aggregates in sarcoidosis samples. Together, our finding suggests that despite their heterogeneity, the formation of tertiary immune structures is a common feature in granulomas from TB patients.

Tuberculosis and diabetes mellitus: Relating immune impact of co-morbidity with challenges in disease management in high burden countries.

Mycobacterium tuberculosis (MTB) is the causative agent of TB. TB incidence is high in many low resource settings where limited health systems make it difficult for screening of co-morbid conditions. Susceptibility to TB is increased with coincident diabetes mellitus (DM) or prediabetes. DM leads to chronic, subclinical inflammation in the host leading to compromised protective immunity against MTB, impacting TB treatment. This review focuses on the immunological impact of DM and prediabetes on TB infections, highlighting the importance of having effective diagnostic, treatment and management programs for early identification of hyperglycemia in TB patients to improve treatment outcomes. Further, it describes challenges in monitoring of TB and DM co-morbidity in a high-burden setting.

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.

Latent M. tuberculosis infection is associated with increased inflammatory cytokine and decreased suppressor of cytokine signalling (SOCS)-3 in the diabetic host.

Tuberculosis (TB) outcomes are worsened by type II diabetes mellitus (DM). Protective immunity against Mycobacterium tuberculosis (MTB) is driven by cytokines. Latent TB (LTBi) is common but its effect on the diabetic host is not well understood. We investigated mycobacterial antigen-stimulated responses in peripheral blood mononuclear cell (PBMC) isolated from healthy endemic controls (EC), those with LTBi, DM groups with and without LTBi, as compared with TB patients. Cytokines were measured using a Luminex-based assay. Gene expression was determined by RT-PCR. In DM-LTBi cases, PPD-stimulated proinflammatory cytokines; IFN-γ, IL-6, IL-2, TNF-α and GM-CSF and anti-inflammatory cytokines, IL-5 and IL-13 were raised as compared with EC. DM-LTBi PPD-stimulated IFN-γ, IL-6 and TNF-α mRNA titres were found raised in DM-LTBi, whilst suppressor of cytokine signalling (SOCS)-3 expression was lowered. Within DM cases, stratification based on HbA1c levels revealed raised IFN-γ but lowered IL-6 gene expression in those with controlled levels as compared with uncontrolled glycaemic levels. Further, SOCS1 expression levels were found higher in DM cases with controlled glycaemia when compared with EC. Overall, we show that diabetics with LTBi manifest raised levels of inflammatory and anti-inflammatory cytokines concomitant with reduced SOCS3 mRNA expression. Reduced glycaemic control results in further inflammatory dysregulation impacting conversing impacting IFN-γ and IL-6 activation. These results suggest that dysregulated immune activation in diabetes is exacerbated by LTBi, lack of glycaemic control may further compromise immunity against MTB infection.

Lipid A-Ara4N as an alternate pathway for (colistin) resistance in Klebsiella pneumonia isolates in Pakistan.

OBJECTIVES: This study aimed to explore mechanism of colistin resistance amongst Klebsiella pneumoniae isolates through plasmid mediated mcr-1 gene in Pakistan. Carbapenem and Colistin resistant K. pneumoniae isolates (n = 34) stored at - 80 °C as part of the Aga Khan University Clinical Laboratory strain bank were randomly selected and subjected to mcr-1 gene PCR. To investigate mechanisms of resistance, other than plasmid mediated mcr-1 gene, whole genome sequencing was performed on 8 clinical isolates, including 6 with colistin resistance (MIC > 4 µg/ml) and 2 with intermediate resistance to colistin (MIC > 2 µg/ml). RESULTS: RT-PCR conducted revealed absence of mcr-1 gene in all isolates tested. Whole genome sequencing results revealed modifications in Lipid A-Ara4N pathway. Modifications in Lipid A-Ara4N pathway were detected in ArnA_ DH/FT, UgdH, ArnC and ArnT genes. Mutation in ArnA_ DH/FT gene were detected in S3, S5, S6 and S7 isolates. UgdH gene modifications were found in all isolates except S3, mutations in ArnC were present in all except S1, S2 and S8 and ArnT were detected in all except S4 and S7. In the absence of known mutations linked with colistin resistance, lipid pathway modifications may possibly explain the phenotype resistance to colistin, but this needs further exploration.

Upregulated type I interferon responses in asymptomatic COVID-19 infection are associated with improved clinical outcome.

Understanding key host protective mechanisms against SARS-CoV-2 infection can help improve treatment modalities for COVID-19. We used a blood transcriptome approach to study biomarkers associated with differing severity of COVID-19, comparing severe and mild Symptomatic disease with Asymptomatic COVID-19 and uninfected Controls. There was suppression of antigen presentation but upregulation of inflammatory and viral mRNA translation associated pathways in Symptomatic as compared with Asymptomatic cases. In severe COVID-19, CD177 a neutrophil marker, was upregulated while interferon stimulated genes (ISGs) were downregulated. Asymptomatic COVID-19 cases displayed upregulation of ISGs and humoral response genes with downregulation of ICAM3 and TLR8. Compared across the COVID-19 disease spectrum, we found type I interferon (IFN) responses to be significantly upregulated (IFNAR2, IRF2BP1, IRF4, MAVS, SAMHD1, TRIM1), or downregulated (SOCS3, IRF2BP2, IRF2BPL) in Asymptomatic as compared with mild and severe COVID-19, with the dysregulation of an increasing number of ISGs associated with progressive disease. These data suggest that initial early responses against SARS-CoV-2 may be effectively controlled by ISGs. Therefore, we hypothesize that treatment with type I interferons in the early stage of COVID-19 may limit disease progression by limiting SARS-CoV-2 in the host.

Higher entropy observed in SARS-CoV-2 genomes from the first COVID-19 wave in Pakistan.

BACKGROUND: We investigated the genome diversity of SARS-CoV-2 associated with the early COVID-19 period to investigate evolution of the virus in Pakistan. MATERIALS AND METHODS: We studied ninety SARS-CoV-2 strains isolated between March and October 2020. Whole genome sequences from our laboratory and available genomes were used to investigate phylogeny, genetic variantion and mutation rates of SARS-CoV-2 strains in Pakistan. Site specific entropy analysis compared mutation rates between strains isolated before and after June 2020. RESULTS: In March, strains belonging to L, S, V and GH clades were observed but by October, only L and GH strains were present. The highest diversity of clades was present in Sindh and Islamabad Capital Territory and the least in Punjab province. Initial introductions of SARS-CoV-2 GH (B.1.255, B.1) and S (A) clades were associated with overseas travelers. Additionally, GH (B.1.255, B.1, B.1.160, B.1.36), L (B, B.6, B.4), V (B.4) and S (A) clades were transmitted locally. SARS-CoV-2 genomes clustered with global strains except for ten which matched Pakistani isolates. RNA substitution rates were estimated at 5.86 x10-4. The most frequent mutations were 5' UTR 241C > T, Spike glycoprotein D614G, RNA dependent RNA polymerase (RdRp) P4715L and Orf3a Q57H. Strains up until June 2020 exhibited an overall higher mean and site-specific entropy as compared with sequences after June. Relative entropy was higher across GH as compared with GR and L clades. More sites were under selection pressure in GH strains but this was not significant for any particular site. CONCLUSIONS: The higher entropy and diversity observed in early pandemic as compared with later strains suggests increasing stability of the genomes in subsequent COVID-19 waves. This would likely lead to the selection of site-specific changes that are advantageous to the virus, as has been currently observed through the pandemic.

Testing for Mycobacterium tuberculosis infection using the QuantiFERON-TB GOLD assay in patients with comorbid conditions in a tertiary care endemic setting.

INTRODUCTION: There were 10 million new cases of tuberculosis (TB) in 2017. To eliminate TB, it is necessary to diagnose active TB and latent tuberculosis infection (LTBI). Diagnosis of paucibacillary disease and in extrapulmonary TB (EPTB) remains challenging; low mycobacterial load can be missed by microbiological or molecular based confirmation; EPTB, can be misdiagnosed due to absence of site specific specimens for testing. Interferon gamma release assays (IGRA) use T cell-based Interferon-gamma (IFN-γ) to identify infection with M. tuberculosis (MTB) but cannot discriminate between active and LTBI. We investigated how IGRA was being used in a high burden low resource setting. METHODS: We conducted a retrospective review of 149 consecutive cases received for QuantiFERON-TB Gold In-Tube Assay (QFT-GIT) testing in routine clinical service. RESULTS: Fifty-six cases were QFT-GIT positive and 93 were QFT-GIT negative. Thirty-six per cent of QFT-GIT tested cases had active TB. Of QFT-GIT positive cases, 59% patients had active TB; 10 with pulmonary and 23 with extra-pulmonary TB. The remaining 41% QFT-positive cases were LTBI. Of the QFT-GIT negative cases, 22% had active TB. Co-morbid conditions were present in 37% of QFT-GIT positive and 60% of QFT-GIT negative cases. CONCLUSIONS: Our study shows that IGRA is being used as an adjunct test for active TB in this population. It highlights the complexity of interpreting QFT-GIT results particularly for QFT-GIT negative cases when ruling out MTB infection.

Role of TNF α, IL-6 and CXCL10 in Dengue disease severity.

BACKGROUND AND OBJECTIVES: Dengue virus infections (Dengue) have become increasingly common in Pakistan and can result in case fatalities if not managed appropriately. Patients with Dengue virus infection may be asymptomatic or present with Dengue fever (DF), Dengue with warning signs (DWS) or severe Dengue (SD). Severity in Dengue is coincident with an exacerbated production of lymphocyte-induced cytokines and chemokines which are associated with plasma leakage. We investigated the association of circulating levels of cytokines such as Interleukin (IL)-6, tumor necrosis factor (TNF)-alpha and CXCL-10 in Dengue patients with differing severity of disease. MATERIALS AND METHODS: Dengue infection was confirmed by testing for human IgM to the Dengue virus. Dengue patients (n=58) and healthy controls (n=33) were recruited. Dengue patients were grouped into those with DF (n=39), DWS (n=15) and SD (n=4). Serum IL-6, TNFα and CXCL10 levels were tested by ELISA. The Mann Whitney U test was used for statistical analysis. RESULTS: Circulating levels of TNFα (p≤0.001) and CXCL10 (p≤0.001) levels were increased in Dengue patients as compared with controls. When patients were stratified for disease severity, it was observed that CXCL10 was increased in DWS as compared to DF (p=0.046). IL-6 levels were increased in patients with SD as compared to those with DWS (p=0.044). TNFα levels were not found to differ between different groups of Dengue patients. CONCLUSION: Raised CXCL10 and TNFα levels were associated with increased clinical severity of Dengue infection and probably increased disease progression due to excessive inflammation and increased vascular changes in the patients.

Differential Early Secreted Antigen Target (ESAT) 6 kDa-induced IFN-γ and SOCS1 expression distinguishes latent and active tuberculosis.

INTRODUCTION: Expression of Suppressor of cytokine signaling (SOCS)-1 molecules is increased in patients with tuberculosis (TB). Early Secreted Antigen Target (ESAT)-6 kDa - induced IFN-γ responses indicate Mycobacterium tuberculosis infection. The effect of ESAT6- stimulation on SOCS1 in the host is not known. METHODOLOGY: Healthy asymptomatic controls had a negative (n = 16) or a positive ( n = 13) tuberculin skin test (TST). ESAT6-induced IFN-γ responses classified these controls as positive (EC ESAT6 IFN-γ (+), n = 5) or negative (EC ESAT6 IFN-γ (-), n = 24) responders. Patients had pulmonary (n = 21) or extra-pulmonary (n = 30) tuberculosis. Peripheral blood cells were stimulated with ESAT6 and mRNA expression of IFN-γ and SOCS1 was determined. RESULTS: ESAT6-induced IFN-γ expression was raised in EC ESAT6 IFN-γ (+) as compared with EC ESAT6 IFN-γ (-), p = 0.019. ESAT6-induced SOCS1 mRNA expression was increased in both pulmonary TB and extra-pulmonary TB patients as compared with both EC groups. ESAT6-induced IFN-γ/SOCS1 mRNA expression ratio was decreased in TB patients as compared with both EC groups. CONCLUSION: M. tuberculosis infection induces increased ESAT6-induced IFN-γ responses in both latent and active TB. Our data shows down-regulation of IFN-γ / SOCS1 expression to be induced only in active TB cases, distinguishing them from healthy individuals likely to have latent TB. A decreasing IFN-γ /SOCS1 ratio may leads to reduced Th1 immunity which contributes to inability of the host to control clinical disease.