Short Communication: HIV-1 Drug Resistance Mutation Analyses of Cameroon-Derived Integrase Sequences

HIV/AIDS remains a health concern that endangers the lives of millions of people worldwide. HIV-1 has three essential enzymes used for its replication, namely, protease, reverse transcriptase, and integrase (IN). HIV IN is a 32 kDa protein encoded at the 3′-end of the HIV pol gene. ¹ IN consists of 288 amino acids, and can be divided into three structural and functional domains: a zinc-binding N-terminal domain, a catalytic core domain, and a DNA binding C-terminal domain. ²

IN strand transfer inhibitors (INSTIs) are a class of antiretroviral drugs used to target the IN enzyme, to prevent viral complementary DNA integration into the host genome. IN resistance-associated mutations (RAMs) located at amino acid residues Y143C, Q148H, and N155H have shown to be the primary/major pathways that induce resistance to the INSTI raltegravir (RAL). Primary mutations that induce resistance against elvitegravir (EVG) include T66I, E92Q, Q148H/R/K, and N155H. RAL and EVG present broad cross-resistance. ³ Dolutegravir (DTG) retains its activity against viruses that exhibited RAMs against RAL and EVG. ⁴ However, combinations of mutations at position G140 and Q148 can result in significant drug resistance to DTG. Furthermore, DTG monotherapy in treated naive patients has been reported to be associated with more frequent development of RAMs such as R263K, G118R, and S230. ⁵

Naturally occurring polymorphisms (NOPs) have been identified at sites associated with secondary RAMs: L74V, M154I, I72V, and T125A. ⁶ Studies have shown that difference across HIV-1 subtypes may play a role, by influencing NOPs in facilitating or compensating the development of major resistance pathway to antiretroviral drugs. ⁷ Owing to the development of drug resistance across currently available drugs, the World Health Organization (WHO) has put forth the use of the INSTI DTG as part of suggested first-line therapy. Therapeutic advantage of DTG is its ability to maintain high potencies against mutant strains of HIV-1 that previously exhibited resistance to other combination antiretroviral therapy (cART) drugs. ⁷ In Cameroon, there is a lack of data regarding the emergence of RAMs against INSTIs; particularity DTG as it is anticipated to be used in future treatment regimens. ⁸ Therefore, it is of paramount importance to identify RAMs from samples sequenced before the initiation of INSTI therapy in Cameroon.

In this study, we evaluated only IN sequences from INSTIs-naive HIV-infected individuals from Cameroon, dating between 1994 and 2009, obtained from the Los Alamos National Library (LANL) HIV-1 database (https://www.hiv.lanl.gov/components/sequence/HIV search.com), accessed between June 10, 2019, and June 12, 2019. We screened for the presence of primary mutations, secondary mutations, and NOPs.

Ethical permission for this study was obtained from the Health Research Ethics Committee of Stellenbosch University (N15/08/071). Our search inclusion criteria included all Cameroonian IN sequences identified from treatment-naive patients. Online-derived sequences used were sampled between 1994 and 2009, before the start of INSTIs in Cameroon. We selected one sequence per patient and 655 HIV-1 sequences were included in the analyses.

For HIV-1 subtyping, the IN sequences were reanalyzed between June and July 2019, using the REGA version 3 online subtyping tool (www.bioafrica.net/rega-genotype/html/subtypinghiv.html), Stanford University HIV Drug Resistance Database (http://hivdb.stanford), and COMET (https://comet.lih.lu/). ⁹

IN nucleotide, sequences, and predicted amino acid sequences were compared with the HXB2 HIV-1 subtype B consensus sequence (GenBank accession no. K03455). Mutations associated with resistance to INSTIs were identified using the Stanford University genotypic resistance interpretation algorithm, HIVdb version 8.3 (http://hivdb.stanford.edu/). Drug resistance mutation results were classified as either major or minor mutations, last accessed July 18, 2019.

Among the 577/655 (88.2%) group M viruses, sequences were classified as belonging to subtypes CRF02_AG (44.7%) n = 293/655, A (12%) n = 79/655, CPX (18.0%) n = 118/655, CRF01_AE (0.6%) n = 4/655, D (1.9%) n = 13/655, F (5.3%) n = 35/655, and G (5.3%) n = 35/655. Sequences from (1.8%) n = 12/655 individuals were classified as belonging to group N, group O (8.7%) n = 57/655, and group U (1.4%) n = 9/655.

In total, 1.4% (9/655) of the database-derived sequences had HIV-1 RAMS: this includes T66A detected in 4/655 (0.6%), R263K detected in 2/655 (0.3%), N155K/T detected in 2/655 (0.3%), Q148V detected in 1/655 (0.2%) and E92S detected in 2/655 (0.3%). Of note, 2/655 (0.3%) of sequences that contained E92S occurred in combination with another mutation, in this case, one sequence had E92S in combination with Q148V and the other sequence had E92S in combination with T66A.

In addition, 66/655 (10.1%) of the sequences contained seven different accessory RAMs, namely, A128T detected in 2/655 (3.0%), D232N detected in 3/655 (4.5%), E157Q detected in 19 (28.7%), G140A detected in 2/655 (3%), Q146R detected in 1/655 (1.5%), Q95K detected in 2/655 (3.0%), and T97A detected in 37/655 (5.6%).

Our findings are in agreement with other reported studies, where CRF02_AG is seen as the predominant subtype causing HIV-1 infection in Cameroon. ^8,10 We further interrogated the IN region for the presence of INSTI RAMs and we identified major and minor RAMs in the sequences investigated. In total, 9/655 (1.4%) of the sequences exhibited major RAMs. Of note, these mutations reduce susceptibility to EVG and RAL. The R263K mutation was detected in our database-derived sequences. It occurs in a high proportion of patients developing virological failure (VF) on an incompletely suppressive DTG-containing regimen and rarely in patients receiving RAL. It reduces DTG and bictegravir susceptibility approximately twofold and reduces EVG susceptibility somewhat more. ¹¹ In addition, Q148H may profoundly affect second-generation INSTI susceptibility. If co-occurring with additional RAMs, mutations in the Q148 pathway can lead to higher fold resistances against all INSTI. The results reported are in line with the previous findings by our laboratory, which reported the circulation of RAMs from cART-naive patients in Cameroon. ⁸

We further detected large number of patients' cohort sequences harboring accessory mutations, with T97A (56.1%) being the most frequent accessory mutations detected, followed by E157Q detected in 28.7%. T97A substitution is a clinical worrisome mutation, since it reduces susceptibility to RAL and DTG, when combined with other major RAMs. E157Q is an accessory mutation usually selected in patients receiving RAL and in vitro by EVG. It has also been reported in a patient with VF on a DTG-containing regimen. It appears to reduce RAL and EVG susceptibility by two- to threefold and to increase DTG susceptibility. ¹² NOPs that are mostly not detected at baseline testing are likely to compensate or delay the development of resistance, according to other reported findings. ¹³

From this study, we observed the occurrence of INSTI RAMs from cART-naive patient's sequences. The occurrence of INSTI RAMs and NOPs among Cameroon-derived IN sequences warrants baseline resistance testing, before cART commencement, as these patients may be at higher risk of failing their INSTI-based therapy.