Development of ELISA and lateral flow test kits for Human Immunodeficiency Virus guided by the prevailing consensus sequence of HIV Env gene in Kenya

Abstract

The Env gene of Human Immunodeficiency Virus (HIV) is vulnerable to mutations that could contribute to variability of diagnostic sensitivity of antibody-based HIV diagnostic tests. Previous studies established that some HIV Enzyme-linked Immunosorbent Assay (ELISA) kits displayed different diagnostic sensitivities when they were used to test blood samples from various geographical locations. Currently, the most widely used testing platforms for HIV in Kenya are the ELISA and Lateral Flow tests (LFTs) which are coated with peptides that have been developed using global consensus sequence of HIV Env gp41 Immunodominant Region (IDR) (and sometimes gp120) that may theoretically not be able to detect some local HIV strains. This study was designed to develop and evaluate LFT and ELISA test kits for HIV 1/2 based on the prevailing consensus sequence of HIV Env gene in Kenya. The study was a Laboratory-Based Experimental Design that involved collection of 200 HIV positive and a similar number of HIV negative blood samples from the four Regional Blood Transfusion Centers (RBTCs) in Kenya over a period of eighteen months. From the HIV positive samples, ribonucleic acid (RNA) was extracted, reverse transcribed and then amplified. The resultant cDNA was sent to Macrogen Europe (Amsterdam, Netherlands) for sequencing. The Consensus sequence of HIV Env gp41 IDR gene established in this study and the global consensus of HIV Env gp41 IDR were sent to LifeTein LLC (Hillsborough, NJ) for production of corresponding bulk peptides. These peptides were used with other commercially available reagents, through a series of optimization experiments, to produce ELISA and LFTs which were evaluated using the 400 characterized HIV panels prepared in this study. The study was approved by KEMRI Ethical Review Committee. Blood samples collected from the NBTCs concealed the identities of the donors. The data was collected and entered in the Excel software. Sequences were analyzed using various bioinformatics' tools that included Shannon-Two Entropy, Los Alamos PhyML 3.0 phylogenetic tree software, “RIP” software for ddetermination of HIV sub-types and recombinants and Phyre2 software for peptide structure prediction, among others. This study established the prevailing consensus sequence of Env gp41 IDR gene in Kenya and also developed two HIV testing kits (LFT and ELISA) using Consensus HIV Env gp41 IDR peptide from this gene. There was 100% sequence similarity in the xxii Cysteine Loop and CTL epitope of HIV Env gp41 IDR peptides between Kenya and global consensus peptide sequences. In general, 67.4% of the amino acid positions in Consensus HIV Env gp41 IDR peptide (Kenya) were highly conserved with the Entropy value of below 0.25 (baseline Entropy). Of the 91 samples that were sequenced the sub-type distribution of HIV was A1 (76.9%), C (6.6%), D (14.3%) and CRF A2.CY.94CY017_41 (2.2%). Samples that were tested in this study consisted of 31.9% “recent” HIV infections. In respect to Vironostika™ Uni-Form II Ag/Ab ELISA the developed LFT had a diagnostic sensitivity (D-SN) of 95.2% (95% CI: 90.3-98.0%) which was the same as that of Aware™ HIV-1/2 BSP LFT and close to the two kits that are currently in the National HIV testing Algorithm in Kenya: First Response™1-2.0 (95.9%, 95% CI: 91.2-97.4%) and KHB Colloidal Gold (95.9%, 95% CI: 91.2-98.5%). The developed LFT had a higher D-SN than the “Tie Breaker” kit in the Algorithm, Uni-Gold™ HIV Test (93.8%, 95% CI: 88.5-97.1). The ELISA test kit developed in this study had a D-SN of 97.2% (95% CI: 93.1-99.2%). The study also established that the overall D-SN of using LFTs in HIV testing in Kenya was 96.0% (95% CI: 92.3-98.3%) in respect to Vironostika™ Uni-Form II Ag/Ab ELISA and that among the LFTs, Determine™ HIV-1/2 showed the highest values of both D-SN and analytical sensitivity, 96.6% (95% CI: 92.2-98.9%) and 63pg/ml respectively, making it the LFT that would have been the most preferred choice of a Screening test in the National HIV testing Algorithm in Kenya were not for its being 10-16% more expensive than the current kits in this Algorithm. This study also established that although there were some variations of the HIV Env gp41 IDR peptide in Kenya in comparison with global consensus of the same, there were no differences in the performance of the kits developed using Consensus HIV Env gp41 r-IDR (Kenya) and Consensus HIV Env gp41 r-IDR (Global) peptides. The study also established that the analytical sensitivity of Determine™ HIV-1/2 Ag/Ab Combo and Vironostika™ Uni-Form II Ag/Ab ELISA in detection of p24 antigen were the same at 63pg/ml. This study recommends the following: return of Determine™ HIV-1/2 to the National HIV Testing Algorithm due to its superior performance, regular monitor of the effectiveness of the HIV testing kits in Kenya for the possible emergence of HIV variants that could escape detection by these kits and seeking of more funds to support the commercialization of the prototype HIV kits developed in this study.