The principal aim of the Federation of African Immunological Societies (FAIS) Legacy Project (FLP) is to increase the human capacity for research in immunology and related sciences on the African continent by training 1000 new PhD students over ten years. See more – faislegacyproject.com
Esemu Livo Forgu is an FLP awardee from Cameroon from the The Biotechnology Centre, University of Yaoundé I. Esemu PhD project is entitled “The effects of HIV-1 infection on microRNA important for the trans-placenta transfer of maternal antibodies”. Esemu chose the Cell Biology Laboratory, National Institute of Communicable Diseases in South Africa under Caroline Tiemessen for his PhD exchange project.
Esemu Livo Forgu Report
Report of activities at the Cell Biology Laboratory, Center for HIV and STI, National Institute of Communicable Diseases, Johannesburg, South Africa
I express my sincere gratitude to the FLP for supporting this exciting and enriching journey of science to South Africa. It was a life changing experience.
The original aims of my research project were to:
1) Measure plasma and placental miRNA levels in HIV infected and uninfected women
The hypothesis behind this was:
1) HIV positive pregnant women will have lower levels of endocytic and FcRn promoting miRNAs compared with HIV negative women.
2) The levels of these miRNAs will correlate with antibody transfer ratio.
To attain this aim, we did intend to measure peripheral plasma and placental tissue homogenate levels of miRNAs (miR-516-5p, miR-517, miR-518b, miR-520h, miR-525, mR-526, miR-150, miR-3181, miR-199a, miR-199b) in HIV positive and HIV negative women by qPCR and correlate these with mom/cord ratios of malaria antibodies.
2) Measure the expression levels of FcRn receptor in placental tissues derived from HIV-infected and uninfected pregnant women at delivery.
Hypothesis: I. FcRn levels are lower in HIV infected women compared to HIV uninfected women. II: FcRn levels will correlate with antibody transfer ratios. III. miRNAs correlate with the FcRn levels.
Our method entailed measuring the FcRn expression levels from excised placenta homogenates of HIV-infected and uninfected women by ELISA.
Samples were shipped to the Cell Biology Laboratory, NICD and after discussing with Prof. Caroline Tiemessen and Dr. Paximadis, the Molecular Biologist of the Cell Biology Laboratory, the primary research objective which aimed to measure plasma and placenta miRNA levels in HIV infected and uninfected individuals was maintained, however, we elected to focus on the placenta given that the role of miRNA in the abnormalties observed in the placenta of HIV positive women is not understood. We also changed the method to be used from qPCR of select miRNAs to whole genome microRNA sequencing of selected placenta biopsies using the NeqSeq New Generation Sequencer (NGS). The primary reason for the change was, given the size of my proposed microRNA panel, using NGS is a more cost-effective option both in the quality and the depth of the information that would be generated. The Sequencing Core Facility (SCF) of NICD was the designated site to run miRNA NGS however, the sequencing instrument was unavailable due to delays in delivery – sequencing will therefore only be done after my departure date from South Africa. As result, the aim of my trip was repurposed to:
1) Perform miRNA extraction of placenta biopsies and ensuring their quality for NGS
2) Shadow a scientist at the Sequencing Core Facility (SCF) to understand he principles of sequencing and miRNA sequencing
3) Learn bioinformatics related to miRNA analyses.
After optimization, we opted for a procedure that would allow the extraction both mRNA and miRNA. The absence of precise and cost-effective technologies to measure the quality of miRNA made us opt for an indirect approach which measures mRNA quality as a proxy for the quality of all total RNA in the samples. miRNA was extracted from 79 placenta biopsies under the watch of Dr. Paximadis. Of the 79 extracts, only 29 met the requirements for downstream analyses as depicted in Table 1. Samples will further be screened for quality using a panel of selected microRNAs (U6, SNORD48, SNORD44, miR-16, and 5S).
Table 1: Summary of sample selection for NGS
|Total number of samples||36||43||79|
|Visually good quality||9||16||25|
To achieve aim 2, I was introduced to Dr. Zamantungwa Khumalo a Molecular Biologist at the SCF who walked me through the process of Next Generation Sequencing with specifications on miRNA. We then had several working sessions to provide me theoretical and visual experience on NGS. We went through library preparation to the purification and size determination of the library, sample preparation, and preparing the equipment for a sequencing run. The concepts of PCR were reiterated as I ran trial experiments which gave me greater mastery and the desire to teach others in Cameroon. It was a great experience and the sessions were run with professionalism however, I did not have the possibility to run samples myself. From experience, observing a process and performing the experiment oneself seem similar but are exceeding disparate. I therefore still lack the hands-on experience in performing NGS.
My last aim was to learn bioinformatics related to analyzing miRNA. Dr. Stanford Kwenda gave me the theory underlying the choice of each tool we will use. We had an introduction to basic commands, he then provided me a mock dataset from which we analyzed using the tools. An important step is to download relevant databases which are all important for performing alignments and filtering. Also, we used the DESeq 2 function in R 3.5.0 to identify differentially expressed genes in our mock dataset.
The plan is for me to perform the bioinformatics analyses of my samples under the guidance of Dr. Kwenda once we get our data from the sequencer.
Understanding “basic” bioinformatics is useful especially in our times of big data analyses. It would require continuous dedication on my part to further grow in this field. The laptop purchased for this trip was a good one but would require an upgrade for the analyses of my data.
At the end of my stay I was privileged to give a presentation at the PRF hall to my newfound colleagues.
I attended prestigious events such as Professorate inaugural lecture of Prof Bavesh Kana, James Gear Memorial lecture (Dr. Melinda Suchard), to name a few and I was a participant at the Infectious Disease Africa Symposium. I was in a video clip introducing Prof Caroline Tiemessen as a winner of the prestigious South African Medical Research Council Scientific Gold Merit Award https://www.youtube.com/watch?v=SXVPKPrBxpM.
Although I could not attain my original objectives, visiting the NICD has rekindled my aspiration to be a leading scientist in my field. This opportunity was unique to me because it gave me the possibility to strengthen the bond between my mentor and I, acquire new skills and I made wonderful friends and acquaintances. The foundation for the collaboration between the Biotechnology Center, University of Yaounde 1, the Institute of Medical Research and Medicinal Plant Studies, Cameroon and the National Institute of Communicable Diseases, South Africa has been laid – on which we could build on in the future.
The next step would require us to identify reference microRNAs in the placenta to complete the selection process for the NGS this December, as well as increase the size of our samples with new sample sets from Cameroon and South Africa. In preparation for an anticipated future publication, I am drafting an introduction to begin the process.
My special appreciation goes to my mentor, Prof Caroline Tiemessen who supports this research through: The South African DSI/NRF Research Chair Initiative (SARChI) grant (Tiemessen; DST/NRF Research Chair of HIV Vaccine Translational Research, University of the Witwatersrand);01/01/2018-12/31/20224
And Prof. Rose Leke for supporting my career as a researcher.