Temporal Trends in SARS-CoV-2 Antibody Levels Among COVID-19 Patients in Kerala During the First Wave and Pre-vaccination Period.

BACKGROUND: The SARS-CoV-2 virus interacts with host cells through the S1 domain of its spike protein. This study measures the IgG immune response to this domain in COVID-19 patients from Kerala, India, and explores its association with various health factors. METHODS: A cohort of 258 COVID-19 patients was analyzed for IgG antibodies targeting the S1 spike protein domain. The temporal pattern of the IgG response and its correlation with hospitalization needs, intensive care, and pre-existing conditions such as diabetes, hypertension, and coronary artery disease were assessed. RESULTS: A significant IgG response (76.4%) was detected, indicating robust immune activation post-infection. The IgG levels peaked between two to four and four to eight weeks post-infection, with a notable increase at 12 weeks, hinting at possible secondary exposure or an immune memory response. No correlation was found between IgG levels and the presence of diabetes mellitus, hypertension, or coronary artery disease. However, higher IgG responses correlated with the severity of the infection, as seen in patients requiring hospitalization or intensive care. CONCLUSIONS: The IgG response to the S1 spike protein domain serves as a potential marker of immune activation in COVID-19. It reflects the body's defense mechanism against the virus and may predict disease severity and outcomes. The findings suggest that IgG levels could be indicative of the viral load, inflammatory response, and possibly the likelihood of protection against reinfection.

High prevalence of "non-pathogenic" POLE mutation with poor prognosis in a cohort of endometrial cancer from South India.

OBJECTIVE: The Cancer Genome Atlas (TCGA) project identified favorable prognosis regarding the ultra-mutated endometrial cancer (EC) subtype linked to polymerase epsilon gene (POLE) mutations. This study investigated POLE mutations in EC of Indian patients. METHODS: This retrospective analytical study was conducted between January 2016 and January 2023 at the Government Medical College, Kozhikode, and the MVR Cancer Center, Kozhikode, Kerala. Sanger sequencing of POLE gene exons 9 and 13 in 151 EC patients was carried out to analyze the relationship between mutations and epidemiological factors, clinicopathologic features, and treatment outcomes. RESULTS: Among 151 cases enrolled, 39 were unique POLE-mutated cases. Significant associations were high-grade tumors, myometrial invasion >50%, and Lymph-vascular space invasion (LVSI). The median follow-up was 40 months (95% confidence interval [CI], 34-46). A lower mean disease-specific survival (DSS) of 51.7 months (95% CI, 43.7-59.6) was noted in the POLE-mutated group compared with 72.11 months (95% CI, 67.60-76.62) for the POLE wild-type. A statistically significant hazard ratio (HR) of 2.683 for DSS in the POLE-mutated group was noted. In advanced stages (FIGO stages II-IV), a nine-fold HR for DSS and overall survival (OS) compared with POLE wild-type was identified. After controlling for treatment effects using Cox proportional HR, advanced-stage POLE-mutated tumors had a significantly higher HR of 8.67 for DSS compared with POLE-wild-type tumors of the same stage. CONCLUSION: This study identified a unique set of POLE mutations in Indian EC patients associated with poor prognosis, which were particularly pronounced in advanced stages. Advanced stage of presentation, type of POLE mutations, and possibly ethnicity are predictors of adverse outcomes in POLE-mutated EC. The present study highlights ethnicity as a determinant of phenotypic expression of genetic change.

Genomic surveillance of SARS-CoV-2 by sequencing the RBD region using Sanger sequencing from North Kerala.

Next Generation Sequencing (NGS) is the gold standard for the detection of new variants of SARS-CoV-2 including those which have immune escape properties, high infectivity, and variable severity. This test is helpful in genomic surveillance, for planning appropriate and timely public health interventions. But labs with NGS facilities are not available in small or medium research settings due to the high cost of setting up such a facility. Transportation of samples from many places to few centers for NGS testing also produces delays due to transportation and sample overload leading in turn to delays in patient management and community interventions. This becomes more important for patients traveling from hotspot regions or those suspected of harboring a new variant. Another major issue is the high cost of NGS-based tests. Thus, it may not be a good option for an economically viable surveillance program requiring immediate result generation and patient follow-up. The current study used a cost-effective facility which can be set up in a common research lab and which is replicable in similar centers with expertise in Sanger nucleotide sequencing. More samples can be processed at a time and can generate the results in a maximum of 2 days (1 day for a 24 h working lab). We analyzed the nucleotide sequence of the Receptor Binding Domain (RBD) region of SARS-CoV-2 by the Sanger sequencing using in-house developed methods. The SARS-CoV-2 variant surveillance was done during the period of March 2021 to May 2022 in the Northern region of Kerala, a state in India with a population of 36.4 million, for implementing appropriate timely interventions. Our findings broadly agree with those from elsewhere in India and other countries during the period.

Clinical, immunological and genomic characteristics of children with X-linked agammaglobulinemia from Kerala, South India.

X-linked agammaglobulinemia (XLA) is an X-linked recessive primary immunodeficiency disorder caused due to a pathogenic variant in the Bruton tyrosine (BTK) gene with an incidence of 1:379,000 live births and 1:190,000 male births. Patients affected with XLA present with recurrent infections of the gastrointestinal and respiratory tracts. Here we report the first case series of 17 XLA patients of 10 South Indian families with a wide spectrum of clinical and genetic features. In our cohort, patients presented mainly with recurrent pneumonia, gastrointestinal infection, otitis media, pyoderma, abscesses, empyema, arthritis, and osteomyelitis. Using next-generation and Sanger sequencing we have identified 10 unique pathogenic and likely pathogenic variants in 17 patients. This encompasses three nonsynonymous, two stop-gain, two frameshifts, two structural, and one splicing variant, out of which two of them are novel. Based on the type of variant, patients had variable clinical features and treatment responses. We have also evaluated Btk protein expression for six patients in comparison to the healthy individuals and determined mosaic Btk expression patterns in four mothers. We have also performed family screening in 6 families using Sanger sequencing and identified 19 carriers for the variant. The diagnosis for the patients led to the proper treatment i.e. 15 patients were on intravenous immunoglobulin (IVIG) and the other two had successful hematopoietic stem cell transplantation (HSCT). Unfortunately, two of our patients died due to sepsis, while on IVIG. We envision the present study could help in better understanding of patients with XLA and help in family screening and prenatal diagnosis. To the best of our knowledge, this is the largest case series of patients affected with XLA from South India.

Spectrum of clinical features and genetic variants in mevalonate kinase (MVK) gene of South Indian families suffering from Hyperimmunoglobulin D Syndrome.

Hyper-IgD syndrome (HIDS, OMIM #260920) is a rare autosomal recessive autoinflammatory disorder caused by pathogenic variants in the mevalonate kinase (MVK) gene. HIDS has an incidence of 1:50,000 to 1:5,000, and is thought to be prevalent mainly in northern Europe. Here, we report a case series of HIDS from India, which includes ten patients from six families who presented with a wide spectrum of clinical features such as recurrent fever, oral ulcers, rash, arthritis, recurrent diarrhea, hepatosplenomegaly, and high immunoglobulin levels. Using whole exome sequencing (WES) and/or Sanger capillary sequencing, we identified five distinct genetic variants in the MVK gene from nine patients belonging to six families. The variants were classified as pathogenic or likely pathogenic as per the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for annotation of sequence variants. Over 70% of patients in the present study had two recurrent mutations in MVK gene i.e. a nonsynonymous variant p.V377I, popularly known as the 'Dutch mutation', along with a splicing variant c.226+2delT in a compound heterozygous form. Identity by descent analysis in two patients with the recurrent variants identified a 6.7 MB long haplotype suggesting a founder effect in the South Indian population. Our analysis suggests that a limited number of variants account for the majority of the patients with HIDS in South India. This has implications in clinical diagnosis, as well as in the development of cost-effective approaches for genetic diagnosis and screening. To our best knowledge, this is the first and most comprehensive case series of clinically and genetically characterized patients with HIDS from India.

Subunit Protein Vaccine Delivery System for Tuberculosis Based on Hepatitis B Virus Core VLP (HBc-VLP) Particles.

Despite the development of modern medicine, tuberculosis (TB), caused by the pathogenic bacterium, Mycobacterium tuberculosis (Mtb), remains one of the deadliest diseases. This bacterium can lay dormant in individuals and get activated when immunity goes down and has also shown considerable prowess in mutating into drug resistant forms. The global emergence of such drug resistant Mtb and the lack of efficacy of Bacille Calmette Guérin (BCG), the only vaccine available so far, have resulted in a situation which cries out for a safe and effective tuberculosis vaccine.Number of different strategies has been used for developing new anti-TB vaccines and several protective antigens have been identified so far. One strategy, the use of protein subunits, has the potential to develop into a powerful tuberculosis vaccine, not only because of its efficacy and safety, but also because they are economical. The proper delivery of protein subunit vaccines with adjuvants or novel delivery systems is necessary for inducing protective immune responses. The available adjuvants or delivery systems are inadequate for generating such a response. In the present method, we have constructed a vaccine delivery system for tuberculosis based on Virus-Like Particles (VLPs). Hepatitis B Virus core antigen gene was recombinantly modified using Overlap Extension PCR (OEPCR). The final construct was designed to express HBc-VLP carrying external antigen (fusion VLP). Mycobacterium tuberculosis antigen CFP-10 was used for the construction of fusion VLP. The recombinant gene for the construct was cloned into a pET expression system and transformed into E. coli BL21(DE3) and induced with IPTG to express the protein. The fusion protein was purified using the Histidine tag and allowed to form VLPs. The preformed VLPs were purified by sucrose density gradient centrifugation. The VLPs were characterized using Transmission Electron Microscopy (TEM).

Vaccine delivery system for tuberculosis based on nano-sized hepatitis B virus core protein particles.

Nano-sized hepatitis B virus core virus-like particles (HBc-VLP) are suitable for uptake by antigen-presenting cells. Mycobacterium tuberculosis antigen culture filtrate protein 10 (CFP-10) is an important vaccine candidate against tuberculosis. The purified antigen shows low immune response without adjuvant and tends to have low protective efficacy. The present study is based on the assumption that expression of these proteins on HBc nanoparticles would provide higher protection when compared to the native antigen alone. The cfp-10 gene was expressed as a fusion on the major immunodominant region of HBc-VLP, and the immune response in Balb/c mice was studied and compared to pure proteins, a mixture of antigens, and fusion protein-VLP, all without using any adjuvant. The humoral, cytokine, and splenocyte cell proliferation responses suggested that the HBc-VLP bearing CFP-10 generated an antigen-specific immune response in a Th1-dependent manner. By virtue of its self-adjuvant nature and ability to form nano-sized particles, HBc-VLPs are an excellent vaccine delivery system for use with subunit protein antigens identified in the course of recent vaccine research.