Cellular, Immunological and Molecular Profiling of HIV Infected Individuals from Pakistan

Abstract

Human Immuno-deficiency virus (HIV) is a serious public health concern since decades. It’s spread across the globe and the diversity of its genome has made it one of the biggest health challenges of our times. Pakistan, earlier classified as the 'low prevalence high risk' country, is now in the 'concentrated phase' of the HIV/AIDS epidemic. Most of the local research has been confined to the genetic aspects of HIV while disease pathogenesis, diagnosis and prognosis have been neglected so far. The present study aimed to explore potential disease severity, disease progression and anti retroviral therapy (ART) response markers along with the comparative analysis of serum microbiome in HIV infection and epidemiological survey of HIV associated co morbidities. A wide array of techniques were utilized including RNA/DNA extraction and analyses, qPCR, RT-PCR, ELISA, flow cytometry and metagenome sequencing to achieve our goals. Epidemiological data was collected from HIV-infected individuals registered at regional HIV treatment centers across the country. HIV was most prevalent among injecting drug users (IDUs) followed by immigrants and male sex workers. The most common co-morbidity was hepatitis C and a significant correlation existed between viral load of HIV infected individuals and Hepatitis C virus (HCV) infected individuals. Elevated levels of CF mt-DNA (Cell free mitochondrial DNA) were observed in individuals infected with HIV, HBV and HCV while healthy individuals showed significantly lower levels of CF mt-DNA suggesting its role in severity of viral infections. Low CD4-positive T-cell counts along with high cell death, elevated CF mt-DNA and caspase-1 expression were observed in untreated HIV infected individuals as compared to anti-retroviral therapy (ART)-receiving and healthy individuals. ART administration improved CD4-positive T-cell counts and lowered caspase-1, CF mt-DNA and cell death. Apoptosis in lymphocytes was higher in xi untreated HIV individuals and significantly reduced upon ART treatment; however, it was still significantly higher than healthy controls indicating the inefficacy of current ART regimes. Direct correlation between CF mt-DNA and cell death along with inverse correlation of CD4-positive T-cells with CF mt-DNA and caspase-1 indicate the potential of CF mt-DNA as a novel disease progression marker. Microbiome alterations are linked to ART response in HIV-infected individuals. Our results indicated significant serum microbiome alterations among healthy, untreated and ART-receiving HIV patients at phylum, class and specie levels. This is the first study from Pakistan that unveils the significance of multiple parameters like CF mt-DNA, caspase-1 and lymphocyte cell death as potential biomarkers of disease severity along with exploration of ART efficacy and alterations in HIV serum microbiome. The work presented here has resulted in the following publications: 1. Ali Z, Waseem S, Shahzadi I, Bukhari S, Anis RA, Ahmed I, Anees M (2021) Association of cell free mitochondrial DNA and caspase-1 expression with disease severity and ARTs efficacy in HIV infection. Molecular Biology Reports, 1-10. 2. Ali Z, Waseem S, Anis RA, Anees M (2020) Assessment of cell free mitochondrial DNA as a biomarker of disease severity in different viral infections. Pakistan Journal of Medical Sciences 36 (5), 860. 3. Ali Z, Shahzadi I, Majeed A, Malik HMT, Waseem S, Ahmed I, Anis RA, Saeed S, Anees M. Comparative analysis of the serum microbiome of HIV infected individuals. Genomics [Submitted]. 4. Ali Z, Shahzadi I, Majeed A, Waseem S, Ahmed I, Anis RA, Saeed S, Akhtar Nasim, Anees M. Epidemiological survey of HIV/HCV coinfected individuals from Pakistan. PloSOne [Submitted].