Impact of INSTI on a Drug-Resistant Mutation (S68S Insertion) in a Patient Infected with HIV-1 CRF06

Abstract

Previously, we reported a T69S insertion in the circulating recombinant form 06_cpx in a patient infected with HIV-1 during the perinatal period. Through this study, we found that the T69S insertion in our previous report was actually an S68S insertion. The patient was treated with zidovudine and didanosine, followed by combination antiretroviral therapy. The introduction of Korean Red Ginseng (KRG) completely suppressed plasma viral RNA to <20 copies/mL and reverted the S68S insertion to wild-type; there was no evidence of an S68S insertion for 3 years. Here, we report the impact of integrase strand transfer inhibitor (INSTI) treatment on drug resistance mutations (DRMs) over a further 10 years. The S68S insertion disappeared after 3 months of INSTI therapy, and the number of DRMs decreased. There were no major DRMs to INSTI in either the patient or her parents. These data highlight the utility of combination therapy with INSTI and KRG.

The emergence of resistance to antiretroviral therapy is a primary cause of treatment failure in persons infected with human immunodeficiency virus type 1 (HIV-1). Resistance to multiple nucleoside analogues may be due to rearrangements in the HIV pol gene, in particular, an insertion at amino acid position 69. The prevalence of the insertion mutation (T69S) in a group of 475 healthy pretreated patients positive for HIV in Spain and in 363 treated patients was four (0.8%) and two (0.5%), respectively. The codon 69 insertion was always found coupled to the T69S mutation.^1,2 In particular, the T69S insertion in HIV-1 reverse transcriptase (RT) confers full resistance to all approved nucleoside reverse transcriptase inhibitors (NRTIs).³ To date, most studies report T69 insertions in subtype B (although there is no such report in Korea) and rarely in non-B subtypes (A, B + F, C, D, F, G, CRF01_AE, CRF02_AG, CRF12_BF, and CRF18_cps) of HIV-1 (https://hivdb.stanford.edu/cgi-bin/InhibitorsMutations.cgi?Gene=RT).

All major HIV treatment guidelines recommend integrase strand transfer inhibitor (INSTI)-based regimens as a first-line therapy for patients infected with HIV-1.⁴ Although INSTI inhibits replication of resistant viruses,⁵ no study has examined the effects of INSTI on the T69S insertion. Recently, we reported that patients treated with combination therapy comprising KRG and antiretroviral therapy (ART) (including INSTI) for approximately 20 years do not develop drug resistance mutations (DRM).⁶

HIV-1 viruses that use the CCR5 coreceptor (i.e., R5 viruses) are generally present over the entire course of infection, whereas those using CXCR4 (i.e., R5X4 or X4 viruses) emerge in approximately 50%–70% of individuals who were infected during the later stages of infection.^7,8 This coreceptor switching is more common in patients who have failed ART.⁹ The false positive rate (FPR) is deﬁned as the probability of erroneously classifying an R5-virus as an X4-virus. In general, the FPR decreases over time,⁹ and no other correlates of tropism switching have yet been identiﬁed.¹⁰

Previously, we reported a T69S insertion in the circulating recombinant form (CRF) 06_cpx of HIV-1.¹¹ Details up to July 2012 are described elsewhere.¹¹ Through this study, we identified that the T69S insertion reported in a previous study¹¹ is in fact an S68S insertion (Supplementary Fig. S1). This error occurred because we relied on genotyping results from the Stanford University HIV Drug Resistance Database (https://hivdb.stanford.edu/hivdb/by-sequences/) and ANRS (https://www.hiv-grade.de/grade_new/), which still classify the sequences as a T69S insertion. To the best of our knowledge, the S68STV insertion is a novel finding. Of note, the patient in this case was administered Korean Red Ginseng (KRG) in addition to ART from May 2007. After 9 months of KRG treatment, the viral load measured in February 2008 was suppressed to <20 copies/mL. The S68S insertion disappeared completely up to August 2010 (Table 1 and Fig. 1).

FIG. 1.

Immunological and virological changes, and changes in the false positive rate (FPR), following treatment with highly active antiretroviral therapy (ART) and Korean Red Ginseng (KRG). Patient A demonstrated S68S insertion in May 2007. Under the same regimen plus KRG, the plasma RNA copy number decreased significantly from 5,500 copies/mL in May 2007 to <20 copies/mL in August 2008, and the S68S insertion disappeared until August 2010.¹¹ Interestingly, the FPR increased during viral suppression and decreased again on reemergence of the S68S insertion. Then, patient A began to take Triumeq from April 2016. S68S insertion disappeared, and the number of drug resistance mutations (DRMs) to PIs and RTIs decreased from eight to three (Table 1). The RNA copy number remained positive due to poor compliance with ART and KRG. None of the three patients developed any major DRMs to integrase strand transfer inhibitors (INSTIs) with the exception of an accessory DRM to INSTI (S153A) in all samples in patient C. Black or crossed and white circles denote the mean positive and negative values for the S68S insertion, respectively. GCT = ART plus KRG; ZDV, zidovudine; ddI, didanosine.

Table 1.

Alignment of Major Resistance Mutations in the Pol Gene, Viral Load and GenBank no. in Three Patients

Date sampled	Months from ART	CD4⁺ T cells	Protease codon			Reverse transcriptase codon									Viral copies/mL	GenBank accession no.
			46	88	90	41	62	67	68		69	70	184	215
			M	N	L	M	A	D	S	ins	T	K	M	T
Patient A	JSE
May 2007	77	1321	ns	ns	M	.	.	.	.	—	.	.	ns	ns	5,500	KC680822
			ns	S	M	.	V	.	.	SV	.	.	ns	ns	5,500	KC680823
May 2007	77	1321	Start of KRG
Aug 2008	92	1018	.	.	.	.	.	.	.	—	.	.	.	.	<20	KC680824
			.	S	M	.	V	.	.	SV	.	.	V	Y	<20	KC680825
			I	S	M	L	.	.	.	—	.	.	V	Y	<20	XX
Feb 2009	98	1279	.	.	.	.	.	.	.	—	.	.	.	.	<20	KC680826
			I	S	M	L	.	.	.	—	.	.	V	Y	<20	KC680827
Aug 2010	116	880	.	.	.	.	.	.	.	—	.	.	.	.	44	XX
			I	S	M	L	.	.	.	—	.	.	V	Y	44	KC680828
Jan 2011	121	742	.	.	.	.	.	.	.	—	.	.	.	.	<20	KC680831
			ns	ns	ns	.	.	.	.	SV	.	.	.	Y	<20	KC680830
			.	S	M	.	.	.	.	SV	.	.	V	Y	<20	KC680829
Jul 2011			Ziagen, 3TC, Reyataz
Sep 2011	129	829	I	S	M	.	V	.	.	SV	.	.	V	Y	100	KC680832
Feb 2012	134	942	I	S	M	.	V	E	.	SV	.	.	V	.	14,500	KC680834
			I	S	M	.	V	E	.	SV	.	.	V	Y	14,500	KC680835
			I	S	M	.	V	E	.	SV	.	.	ns	ns	14,500	KC680833
			ns	ns	ns	ns	V	.	.	SV	.	.	V	Y	14,500	KC680836
Jul 2012	139	762	I	S	M	.	V	E	.	SV	.	.	V	Y	23,200	KC680838
Jul 2013	151	1125	I	S	M	.	V	.	.	SV	.	.	V	Y	<20	KX692410
Jul 2013			Kivexa, Reyataz
Jan 2014	157	983	I	S	M	.	V	E	.	SV	.	.	V	Y	2,580	KX692413
			.	S	M	L	V	.	.	TV	C	.	V	Y	2,580	KX692414
Jun 2014			Kivexa, Kaletra
Jan 2015	169	887	I	S	M	.	V	E	.	SV	.	.	V	C	678	KX692415
Jan 2016	181	643	I	S	M	.	V	E	.	SV	.	.	V	C	993	KX692416
Apr 2016			Triumeq
Jul 2016	187	678	ns	S	M	L	.	.	.	—	.	.	V	C	99,861	PQ032248
			ns	ns	M	.	.	.	.	—	.	.	.	C	99,861	15315
Jan 2017	193	553	I	S	M	.	.	.	.	—	.	.	.	C	9,420	MH054686
			I	S	M	.	.	.	.	—	.	.	.	C	9,420	MH054685
Jan 2017			Stribild
Jul 2017	199	297	.	S	M	.	.	.	.	SS	.	.	V	Y	5,489	MH054688
			I	S	M	.	.	.	.	—	.	.	.	C	5,489	MH054689
			I	S	M	.	.	.	.	—	.	.	.	C	5,489	MH054687
Jan 2018	205	411	I	G	M	.	.	.	.	—	.	.	.	C	45,953	MH054690
			I	G	.	.	.	.	.	—	.	.	.	C	45,953	MH054691
			I	G	M	.	.	.	.	—	.	.	.	C	45,953	15958
Aug 2020	236	200	I	G	.	.	.	.	G	—	.	.	V	C	positive	MW660516
			I	G	.	.	.	.	G	—	.	.	.	C	positive	MW660517
			I	G	.	.	.	.	G	—	.	.	.	C	positive	MW660518
Jul 2021	247	499	I	G	.	.	.	.	G	—	.	.	V	C	positive	OK490552
			.	S	.	L	.	.	G	—	.	.	V	Y	positive	OK490553
			I	G	.	.	.	.	G	—	.	.	.	C	positive	OK490554
Sep 2021			Bictarvy
May 2022	257	408	ns	G	.	.	.	.	G	—	.	.	.	C	positive	PP176138-39
			L	G	.	.	.	.	G	—	.	.	.	C	positive	PP176140-41
Patient B	JIG	Father of patient A
Oct 2019		Genvoya
Dec 2019	241	959	.	.	.	.	.	.	.	—	.	.	.	.	<20	MW660511
Aug 2020	249	1174	.	.	.	.	.	.	.	—	.	.	.	.	<20	MW660513-4
Jul 2021	260	1154	.	.	.	.	.	.	.	—	.	.	.	.	<20	OK490555-6
Patient C	YYH	Mother of patient A
Jun 2013	52	Isentress, Prezista, Norvir
Jul 2013	53	249	.	.	.	.	.	.	.	—	.	.	.	.	82	KX692673
Jan 2014	59	473	Isentress, Kaletra												<20	KX692674
Jan 2015	71	483	.	.	.	.	.	.	.	—	.	.	.	.	<20	KX692675
Jul 2016	89	428	.	.	.	.	.	.	.	—	.	.	.	.	184	MH054696-8
Jan 2018	107	276	Genvoya												<20
Aug 2020	126	679	.	.	.	.	.	.	.	—	.	.	.	.	<20	MW660523-4
Jul 2021	137	425	.	.	.	.	.	.	.	—	.	.	.	.	<20	OK490557-9
Sep 2021			Bictarvy
May 2022	147	278	.	.	.	.	.	.	.	—	.	.	.	.	Positive	PQ032221-6

Hyphen in column ins denotes wild-type without insertion. Dots indicate sequence identity with the wild-type. ns; not sequenced. An accessory mutation (S153A) to INSTI was detected from baseline in patient C. ART, antiretroviral therapy; S68G is not considered to be a resistant mutation.

After the introduction of INSTIs in April 2016, DRMs, including the S68S insertion in the RT, disappeared completely up to May 2022, with the exception of a single amplicon detected in July 2017 (MH054688). In the present study, the S68S insertion disappeared 3 months after INSTI treatment (Table 1), although plasma viremia was not well controlled due to poor compliance. Differing from a previous study reporting the effect of KRG on reducing the plasma RNA copy number to undetectable levels until January 2011,¹¹ the data reported herein suggest that the disappearance of the S68S insertion from more resistant viruses may be due to the potent effect of INSTIs rather than that of KRG. The reason for this is that KRG had been administered continuously since May 2007 (total amount >6,426 g); indeed, disappearance of the S68S insertion has been observed after triple ART and before the introduction of INSTI.⁵ In addition to the disappearance of the S68S insertion, we also noted that the S68S-SV insertion changed into S68S-SS in July 2017 (Table 1). Interestingly, N88G replaced N88S from January 2018. Despite poor compliance by patient A, no major DRM in response to INSTI was detected for 73 months (Fig. 1). In addition, the number of DRMs to protease inhibitors and NRTIs detected in the latest sample decreased from eight to three (Table 1). This is supported by the following: second-generation INSTIs can inhibit resistant forms of integrase,¹² and KRG inhibits HIV-1 replication by slowing coreceptor switching.¹³

Patient B was treated with Genvoya from October 2019 and Bictarvy from 2023, and he showed good compliance with KRG therapy from May 2007 (Fig. 1). He had a history of hypersensitivity to Abacavir because he harbors HLA B57:01. Patient C was treated with INSTI from July 2013 and also showed good compliance with KRG from June 2013 (Fig. 1 and Table 1). No major DRM was detected in either of the parents, despite treatment with GCT for 22 and 13 years, respectively (Table 1).

Their env genes were ampliﬁed by nested PCR or RT-PCR and subjected to direct sequencing.¹³ Tropism was determined using a 10% false positive rate (FPR) cutoff (https://coreceptor.geno2pheno.org/index.php). All were R5 viruses (X4 viruses were detected only in one of three amplicons in July 2013; mean 11.1% = 17.9%, 12.8%, and 2.5% in patient A). The mean FPR in all three patients taking KRG before INSTI therapy gradually increased (Fig. 1) when compared with baseline values, except during a period of re-emergence of S68S-SV (Fig. 1). In contrast, a previous study reported that 2 of 18 hemophiliacs showed an increase in the FPR during ART or GCT therapy (3/3 vs. 2/18, respectively; p < .01).¹³

We collected peripheral blood mononuclear cells at 6-month intervals. Amplification of the full-length pol and env genes was performed as described previously^11
–13 using primer sets targeting the env gene: set 1 = OWE1-JSE(5'-TGGAAKCAGCCAGGAAGTC-3')/594-JSE (5'-TGGTGCTAYAAGCTGGTAC-3'); and set 2 = OWE3-A (5′-AGCMATAGTTGTGTGGACTMTAG-3′)/OWE4-JSE (5'-CAGTCASACCTCAGGTRC-3'). The number of amplicons for the pol and env genes detected in patients A, B, and C was 15 and 10, 7 and 7, 21 and 11, respectively. The plasma RNA copy number was measured using a Roche Cobas Ampli-Prep/Cobas TaqMan HIV-1 kit (version 2.0; Roche Diagnostics Systems, Branchburg, NJ).

These data suggest that INSTI therapy plays a significant role in the disappearance of the S68S insertion,^6,11,14 and that KRG intake plays a role in the increase in FPR as well as in the delayed development of DRM to INSTI.^6,11,12

Footnotes

Sequence Data

The GenBank accession numbers are as follows; KC680822-860, KX692410–416, KX692670-675, MH054682-699, MW660511-24, and OK490555-59 for the pol gene and OR327320-327, OR327358-366, and PQ809491-507 for the env gene.

Authors’ Contributions

Y.K.: Conceptualization; formal analysis; writing—original draft; editing. J.N.: Conceptualization; visualization; methodology; formal analysis; and writing—review and editing.

Author Disclosure Statement

The authors declare no competing financial interests.

Funding Information

This study was supported by a grant from the Korean Society of Ginseng (2022).

Supplementary Material

Supplementary Figure S1

References

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Supplementary Material

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Impact of INSTI on a Drug-Resistant Mutation (S68S Insertion) in a Patient Infected with HIV-1 CRF06_cpx

Abstract

Footnotes

Sequence Data

Authors’ Contributions

Author Disclosure Statement

Funding Information

Supplementary Material

References

Supplementary Material