HIV-1 gp41 genetic diversity and enfuvirtide resistance-associated mutations among enfuvirtide-naïve patients in southern China

doi:10.21203/rs.2.20490/v1

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HIV-1 gp41 genetic diversity and enfuvirtide resistance-associated mutations among enfuvirtide-naïve patients in southern China

https://doi.org/10.21203/rs.2.20490/v1

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Background: Human immunodeficiency virus (HIV) increasing molecular diversity and emergence of drug resistant mutants remain a major concern in Southern China, enfuvirtide (ENF/T-20) is the first entry inhibitor used in patients failing highly active antiretroviral therapy (HAART). However, data on HIV-1 gp41genetic diversity and primary ENF resistance-associated mutations among treatment-naïve patients in southern China is limited. The objective was to identify molecular diversity and ENF resistance patterns of HIV-1 in southern China, using envelop (env) gp41 sequences and bioinformatics tools, which may help optimize ART.

Methods: From December 2018 to January 2019, 439 blood plasma samples from ENF-naïve HIV-1 patients were collected from Shenzhen, Wuhan and Chongqing, of which, 396 HIV env regions were sequenced and subtyped, and were performed the analysis of drug resistance-associated mutations (DRMs).

Results: The main subtypes were circulating recombinant form (CRF) 01_AE (30.6%) and CRF07_BC (48.7%), CRF55_01B had been the fourth subtype in our work, many rare CRFs were observed. Notably, CRF02_AG and CRF_BF strains typically found in Africa and US respectively were identified amongst Chinese HIV-1 patients. Known DRMs were detected in 27.5% (109/396) of ENF treatment-naïve patients. One major DRM (L44M), many secondary DRMs including (N126K, E137K, S138A) and lots of polymorphisms were found in the study, which have been proved to elevate resistance to ENF.

Conclusion: HIV-1 molecular diversity among people in southern China observed in the work indicates that HIV-1 variability is becoming increasingly complex in the south of China. A diverse set of primary DRMs discovered in this study described the serious threat to ART, which remind us the urgent need of timely surveillance of HIV-1 viral diversity and drug resistance in China.

Infectious Diseases

Human immunodeficiency virus (HIV)

Molecular diversity

Subtypes

Enfuvirtide

drug resistance-associated mutations

Human immunodeficiency virus type one (HIV-1) presents high genetic diversity, and is suppressed by antiretroviral therapy (ART), but may acquire many drug resistance-associated mutations (DRMs). HIV-1 genetic studies have revealed four groups (M, N, O and P). The M group of HIV-1 is classified into nine phylogenetic subtypes (A, B, C, D, F, G, H, J and K) and at least 98 circulating recombinant forms (CRFs) [1, 2]. Viral diversity analysis has significant implication for investigating the global origin and evolution of HIV-1 isolates [3], and future vaccine development [4]. Molecular epidemiological studies demonstrated that CRF07_BC, CRF01_AE and CRF08_BC were the dominant subtypes among the population of southern China, with identification of drug-resistant strains and novel unique recombinant forms (URFs) [5–7].

In 2003, Chinese government made it possible to have free access to ART [8], which not included enfuvirtide (ENF). ENF was the first HIV fusion inhibitor based on the amino acid sequence of the heptad repeat-2 (HR2) in the glycoprotein 41 (gp41) of HIV-1 envelope (env) protein, which blocks HIV-1 entry and restrict HIV-1 replication, used in combination with other antiretroviral drugs as an alternative to the ordinary ART [9, 10]. HR1 region substitutions in HIV-1 env gp41, including A30V, L33V, L34M, G36D/S, I37V, V38A, Q39H/R, Q40H, N42T/D, N43D, L44M, L45M, R46M, L54M and et al, have been proved to be associated with ENF resistance in vitro and vivo [11–16]. The major ENF resistance-associated mutations were identified in amino acid position 36–45 of HR1 domain. Other secondary mutations including N126K, E137K, S138A, N140I also were demonstrated to reduce ENF susceptibility [17–20]. ENF resistance-associated mutations have been reported in patients who have accepted highly active antiretroviral therapy (HAART), but less data about the ENF resistance associated mutations among ENF-naïve patients. Hence, before ENF is approved for routine ART by authority in China, the information about ENF primary drug resistance is required.

In this study, we investigated HIV-1 gp41 genetic diversity and the prevalence of antiretroviral drug resistance associated with ENF among treatment-naïve population from southern China.

Study samples

From December 2018 to January 2019, 439 blood plasma samples from ENF treatment-naïve HIV-1 population were collected from the Third People’s Hospital of Shenzhen (228 samples), Wuhan Jinyintan Hospital (44 samples) and Chongqing Centers for Disease Control and Prevention (CDC) (167 samples), and quantitated by HIV-1 nucleic acid detection kit (Zhuhai Livzon Diagnostics Inc., Zhuhai, China). Among these samples, 396 HIV env regions were successfully sequenced.

RNA extraction, amplification and sequencing

HIV-1 RNA was extracted from 650 µL plasma using extraction reagent of HIV-1 nucleic acid detection kit (Zhuhai Livzon Diagnostics Inc., Zhuhai, China) according to the manufacturer’s protocol. HIV-1 env gp41 (HXB2: 7648-8365) was amplified with 5×Neoscript RT Premix (RT-PCR) (Zhuhai Biori Biotechnology Co., Ltd, Zhuhai, China). We used the primers env F-1 5’- TRARGGACAATTGGAGAAGTG-3’; env R-1 5’-GTGAGTATCCCTGCCTAAC-3’. The amplification of the env fragment was performed at 50℃ for 30 min for reverse transcription and then 95℃ for 15 min, followed by 50 cycles at 94℃ for 15 s, 50℃for 30 s, and 72℃ for 1 min, with a final extension at 72℃ for 7 min. The PCR products were purified and sequenced by Invitrogen (Shanghai) trading Co., Ltd using Sanger methods.

HIV-1 genotype and phylogenetic analysis

We edited and aligned the sequences using Geneious 9.1.2, compared with gp41 reference sequences. Env sequences were submitted to the Los Alamos HIV BLAST tool for initial HIV-1 subtyping (https://www.hiv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.html). All sequences were aligned and compared with HIV-1 reference sequences (Accession numbers were shown in supplementary materials S-Table 1) derived from the Los Alamos HIV sequence database (https://www.hiv.lanl.gov) and then the nucleotide alignments were subjected to phylogenetic inference for further HIV-1 subtyping through the neighbor-joining method and Kimura 2-parameter model in 1000 bootstrap replicates implemented in the MEGA 7.0.2 (https://www.megasoftware.net). For finalization of phylogenetic trees, we used FigTree v1.4.3 (University of Edinburgh, UK) to prepare figures as previously described [21]. Intra-genomic breakpoints and boot-scanning analyses were performed on sequences with possibly unidentified strains through jpHMM program (http://jphmm.gobics.de/submission_hiv.html) [22]. The sequences did not match established CRFs were identified as URFs. Recombinant HIV-1 Drawing Tool (https://www.hiv.lanl.gov/content/sequence/DRAW_CRF/recom_mapper.html) was used to generate the genome maps of URFs.

Drug resistance mutation analysis

DRM analyses in env regions were performed in the HIVfird program (https://www.hivfird.ics.ufba.br/). HIVfird uses HIV-1 HXB2 env gp41 protein sequence (nucleotide: 7863-7892, amino acids: 36-45) as a reference sequence to align the user query sequences [23]. The mutation in this region can confer HIV-1 resistance to ENF [24], and polymorphisms are other amino acid differences amongst the gp41 region in the absence of ENF [25].

Statistical analysis

Polymorphisms were analyzed using VESPA program (https://www.hiv.lanl.gov/content/sequence/VESPA/vespa.html). The VESPA program detects signature patterns (atypical amino acid or nucleotide residues) in a set of query sequences relative to a set of background sequences [26]. Chin-square in SPSS 21.0 software was utilized for statistical analyses.

HIV-1 gp41 subtyping analysis

Among 439 samples, a total of 396 samples were successfully sequenced and included in this study, of which 198, 156, 42 were collected from Shenzhen, Chongqing and Wuhan respectively. Gp41 of HIV-1 variant were sequenced and subtyped through jpHMM program and HIV-1 BLAST tool, further confirmed by phylogenetic analysis (Fig. 1). As is shown in Table 1, CRF07_BC (48.7%, 193/396) and CRF01_AE (30.6%, 121/396) were the two main subtypes. A diverse set of genotypes were also identified amongst gp41 regions, including CRF08_BC, CRF55_01B, B, CRF67_01B, CRF_BF, CRF02_AG, CRF52_01B, CRF59_01B, CRF79_0107 and CRF85_BC. Few URFs (0.5%, 2/396) were found in the study (Fig. 2). The distribution of HIV-1 subtypes among the three cities in the study demonstrated that the HIV-1 isolates in Shenzhen were more variable than Chongqing and Wuhan (P < 0.01), and the proportions of CRF07_BC and CRF08_BC in Chongqing were higher than the other two cities (P < 0.01). CRF55_01B outnumbered subtype B to become the fourth HIV-1subtype.

Table 1

Gp41 genetic characteristics of HIV-1 infected populations
Subtypes	Shenzhen (198)	Chongqing (156)	Wuhan (42)	Total N = 396
CRF01_AE	82 (41.4%)	19 (12.2%)	20 (47.6%)	121 (30.6%)
CRF07_BC	78 (39.4%)	101 (64.7%)	14 (33.3%)	193 (48.7%)
CRF08_BC	8 (4.0%)	27 (17.3%)	2 (4.8%)	37 (9.3%)
CRF55_01B	16 (8.1%)	3 (1.9%)	3 (7.1%)	22 (5.6%)
B	4 (2.0%)	4 (2.6%)	3 (7.1%)	11 (2.8%)
CRF67_01B	4 (2.0%)	-	-	4 (1.0%)
CRF_BF	1 (0.5%)	-	-	1 (0.3%)
CRF02_AG	1 (0.5%)	-	-	1 (0.3%)
CRF52_01B	-	1 (0.6%)	-	1 (0.3%)
CRF59_01B	1 (0.5%)	-	-	1 (0.3%)
CRF79_0107	1 (0.5%)	-	-	1 (0.3%)
CRF85_BC	-	1 (0.6%)	-	1 (0.3%)
URF	2 (1.0%)	-	-	2 (0.5%)

Table 2

The characterization of HIV-1 strains with ENF resistance-associated mutations
Code	Source	Subtype	Mutation	Code	Source	Subtype	Mutation
57	CQ	07BC	S35F	10935	CQ	08BC	R46K, S138A
56	CQ	07BC	S138A	10934	CQ	07BC	R46K
120	CQ	55_01B	H53R	10897	CQ	07BC	R46Q, E49D
11665	CQ	08BC	R46Q	10819	CQ	08BC	S35A
11660	CQ	07BC	R46K	10808	CQ	07BC	L33V
11644	CQ	01AE	E137K	10765	CQ	07BC	R46M, E49Q, S138A
11636-2	CQ	07BC	Q32L, R46Q	10611	CQ	08BC	R46Q
11625	CQ	08BC	L33V	10579	CQ	B	Q32L, L44M
11607	CQ	07BC	A30V	10490	CQ	07BC	R46K
11606	CQ	07BC	R46K	10452	CQ	07BC	S138A
11592	CQ	01AE	E137K	10444	CQ	07BC	R46K
11590	CQ	07BC	R46K	10410	CQ	07BC	R46K
11587	CQ	08BC	R46K	10407	CQ	07BC	R46K
11550	CQ	08BC	R46K	10406	CQ	07BC	R46K
11548	CQ	07BC	R46M	S9	SZ	01AE	E137K
11472	CQ	07BC	A30V	S8	SZ	07BC	S138A
11459	CQ	07BC	R46K	S7	SZ	07BC	S138A
114	CQ	07BC	R46K	S5-1	SZ	07BC	R46K
11294	CQ	07BC	S138A	S4-6	SZ	07BC	N126K
11223	CQ	07BC	R46K	S4-36	SZ	01AE	H53Q
11219	CQ	07BC	R46K	S4-30	SZ	01AE	S35F
11190	CQ	07BC	R46K	S4-13	SZ	07BC	R46K
11160	CQ	08BC	S35F, R46K	S4-12	SZ	07BC	S138A
11152	CQ	07BC	R46K	S4-1	SZ	07BC	E137K
11117	CQ	08BC	L33V, R46K, H53R	104 − 40	SZ	01AE	Q56K
11116	CQ	07BC	R46K	S3-4	SZ	08BC	R46K, S138A
11100	CQ	07BC	Q52H	S3-29	SZ	B	Q32L, E137K
11092	CQ	07BC	R46K	S3-23	SZ	01AE	E137K
11091	CQ	07BC	E137K, S138A	S3-20	SZ	07BC	E49Q
11089	CQ	07BC	R46Q	S3-2	SZ	BF1	E49D
11086	CQ	07BC	R46K	S32	SZ	01AE	L34M, S35T
11080	CQ	07BC	R46K, H53R	S2-9	SZ	02AG	R46K, Q56R, E137K
11069	CQ	07BC	R46K	S2-43	SZ	01AE	E137K
11067	CQ	B	H53Q	S2-39	SZ	07BC	S138A
11034	CQ	B	R46M, E137K	S2-29	SZ	07BC	R46K, S138A
11012	CQ	08BC	R46K	S2-28	SZ	08BC	S35F
10939	CQ	07BC	R46K	S2-19	SZ	01AE	E137K
10938	CQ	07BC	R46K	S2-13	SZ	07BC	R46K
10936	CQ	07BC	S35F	S20	SZ	01AE	S35A, H53R
3–4	SZ	59_01B	E137K	S16	SZ	07BC	S35F
2–8	SZ	B	E137K	S15	SZ	01AE	Q52H
2–7	SZ	67_01B	Q52H	452	WH	07BC	H53N
24	SZ	07BC	R46K	3497	WH	B	E137K
2–12	SZ	07BC	R46K, H53R	3493	WH	01AE	Q52H, H53R
2	SZ	01AE	Q52H	3437	WH	01AE	Q39H
18	SZ	07BC	S138A	3387	WH	01AE	E137K
104 − 42	SZ	07BC	R46K	3382	WH	07BC	R46K
104 − 41	SZ	01AE	R46K	3381	WH	B	R46Q
104 − 31	SZ	07BC	Q56K	3370	WH	01AE	E49K
104 − 24	SZ	01AE	Q56R, E137K	3353	WH	07BC	R46K
104 − 22	SZ	07BC	R46K	3350	WH	01AE	E137K
104 − 13	SZ	01AE	L34M, I62V	3247	WH	01AE	S35A, S138A
1	SZ	07BC	S35T	165	WH	07BC	R46K
39	SZ	55_01B	H53R	115	WH	01AE	Q56R, E137K
38	SZ	01AE	E40Q
SZ: Third People’s Hospital of Shenzhen, WH: Wuhan Jinyintan Hospital, CQ: Chongqing CDC.

ENF resistance-associated mutations and polymorphisms in env gp41 regions

In total, 109 (27.5%, 109/396) HIV-1 isolates had ENF resistance-associated mutations and polymorphisms, of which 22 (20.2%, 22/109) had two different substitutions and 3 HIV-1 strains harbored 3 combined mutations. The prevalence of DRMs and polymorphisms among ENF-untreated individuals from Chongqing, Shenzhen, and Wuhan was 34.0% (53/156), 21.7% (43/198) and 31.0% (13/42), respectively. Only one major ENF resistance-associated mutation (L44M) in amino acid position 36–45 of HR1 domain was seen in the enrolled samples. 8.6% (34/396) HIV-1 strains carried secondary DRMs including N126K (1/396, 0.25%), E137K (18/396, 4.55%), S138A (14/396, 3.54%).

Among the 396 ENF treatment-naïve patients, various of polymorphisms were observed in env gp41, R46K/M/Q, E137K and S138A were the major polymorphisms (Fig. 3), R46K/M/Q (19.7%, 38/193) and S138A (5.7%, 11/193) were predominant in subtype CRF07_BC, and E137K (36.4%, 4/11) was a majority in subtype B (P < 0.05), after excluding the subtypes with the number less than ten.

To date, this is the first study on the prevalence of ENF resistance-associated polymorphisms and mutations in HIV-1 isolates among treatment-naïve patients in southern China. What’s more, we investigated the env gp41 diversity of HIV-1 in these patients.

Molecular characterization of the HIV-1 isolates circulating within the HIV-1 patients in southern China revealed the most prevalent subtypes were CRF01_AE and CRF07_BC, while CRF08_BC and subtype B showed the lower prevalence in comparison to the national report in China in 2006 [27]. The prevalence of subtype B mainly transmitted by blood transfusion has significantly decreased, due to the prohibition of paid blood donation, which is demonstrated by the low percentage of subtype B in the present study. The absence of HIV-1 patients from Guizhou, Yunnan and Sichuan provinces in this study may account for the observed low proportion of CRF08_BC, since CRF08_BC predominates in these areas [27]. The geographic subtype distribution (Numbers of samples ≥ 10) suggested that CRF07_BC and CRF01_AE were dominant subtypes in three areas, which was consistent with previous reports among high-risk populations [28–30]. Notably, high prevalence of CRF55_01B was observed in our study, compared with the previous reports [27, 29, 31]. CRF55_01B firstly identified in Shenyang composed of subtype B and CRF01_AE, with 4 intra-genomic breakpoints in the polymerase (pol) gene [32]. Evidence showed that CRF55_01B originated and outbroke from men who have sex with men (MSM) in Shenzhen [33], and CRF55_01B have spread throughout China, along with population migration [34]. Furthermore, several rare CRFs including CRF52_01B, CRF59_1B, CRF67_01B, CRF79_0107 and CRF85 were isolated from HIV-1 patients, describing that HIV-1 recombination between different genotypes is becoming increasingly complex in China.

Significantly, CRF_BF and CRF02_AG strains typically found in South American countries and Africa respectively, were identified among Chinese HIV-1 patients [35–38]. To our knowledge, CRF_BF was firstly reported in China. More importantly, the two uncommon strains were both found in Shenzhen, which was an international metropolis with progressively international travel and immigration. Therefore, the identification of imported CRF_BF and CRF02_AG in Chinese indicates the newly emerging migration modes in the global HIV-1 pandemic. Furthermore, Few URFs were available in our study, HIV recombination can be presented in different regions (gag, pol and env) between different subtypes, therefore, more gene regions should be included in the further study for comprehensive and accurate HIV-1 classification.

In this study, we evaluated the distributions of env gp41 polymorphisms and ENF resistance-associated mutations among HIV-1 strains from 396 ENF-naïve HIV-1 infected population in Shenzhen, Chongqing and Wuhan in the period 2018–2019. In total, 27.5% HIV-1 isolates had primary ENF resistance-associated mutations and polymorphisms, higher than a previous study in Hong Kong (20.8%), China [39]. Only one major ENF resistance-associated mutation (amino acid positions: 36–45), L44M, was discovered in the study, which has associated with 1.8 fold resistance to enfuvirtide in vitro [40]. In China, ENF has been applied as salvage therapy for patients failing ordinary ART since 2015, but not been included in the Free AIDS Antiretroviral Therapy Manual [8], the low usage of ENF in China may contribute to the observed low prevalence of major ENF resistance mutations in our study (0.25%, 1/396), compared to the reports in Brazil (3.8%, 3/80) [19], Spain (3.0%, 6/200) [41] and US (16.7%, 2/12) [42] among ENF-untreated patients. However, several mutations (N126K, E137K, S138A) in the HR2 region presented in the study can lead to increased viral fusion activity and reduced susceptibility to ENF [43]. Notably, these mutations in HR2 domain may dramatically reduce ENF susceptibility about 100-1000-fold, in combination with substitutions in HR1 domain (amino acid positions: 36–45) [43, 44]. In addition, a lot of minor mutations and polymorphisms (A30V, Q32L, L33V, L34M, S35A/F/T, Q39H, R46K/M/Q, E49D/K/Q, Q52H, H53N/Q/R, Q56K/R, I62V) associated with potential resistance ENF were discovered in our study have reported in previous studies [16, 25, 45, 46].

The subtype distribution of three dominant mutations indicated R46K/M/Q and S138A were predominant in subtype CRF07_BC, while E137K was mainly found in subtype B. The prevalence of resistance-associated mutations and polymorphisms may have relationship with HIV-1 subtypes. However, DRM determinations were analyzed by HIVfird Program, which is based on subtype B and has biased the results for non-B strains [23], further phenotypic resistance analysis should be performed to confirm the susceptibility to ENF among non-B subtypes.

In summary, our study demonstrated increasing HIV-1 genetic diversity and high prevalence of primary ENF resistance-associated mutations and polymorphisms in the south Chinese population. The identification of imported CRF02_AG and CRF_BF strains in Chinese individuals, especially the first emergence of US-derived CRF_BF isolate, suggest the newly emerging migration modes of the global HIV-1 pandemic. Additionally, though few major DRM was observed, a large amount of secondary and minor DRMs were isolated from ENF treatment-naïve patients, which can be a serious threat to ART after the failure of the first-line ART. The complex HIV-1 evolution and high prevalence of DRMs to ENF remind us the urgent need for continued molecular surveillance to monitor the HIV-1 diversity and primary DRMs in Chinese population.

HIV: human immunodeficiency virus; ENF: enfuvirtide; HAART: highly active antiretroviral therapy; env: envelope; DRMs: drug resistance-associated mutations; CRFs: circulating recombinant forms; URFs: unique recombinant forms; HR: heptad repeat; pol: polymerase

Data Availability

The data used to support this study are available from the corresponding author upon request.

Ethics approval and consent to participate

The study was approved by the institutional review boards in Third People’s Hospital of Shenzhen, Wuhan Jinyintan Hospital and Chongqing CDC respectively. The methods in the study were in accordance with the guidelines of the Declaration of Helsinki. Written informed consent was obtained from all subjects participating in this research.

Consent for publication

Not applicable.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ contribution

LC and JZ contributed equally to this study. LW, JZ and LC and designed the study. JZ, LC and FG conducted the laboratory tests. JZ, HJ, LZ and XJ collected and analyzed data and prepared the manuscript. LW and LC edited and reviewed the manuscript. All Authors critically reviewed and revised the manuscript drafts, approved the final version of the manuscript and take responsibility for the integrity of the data and accuracy of data analysis.

Funding Statement

This work was supported by grants from National Science and Technology Major Project of the Ministry of Science and Technology of China (2018ZX10306412-004 and 2018ZX10732101-003-002).

Author details

¹National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology; Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, P. R. China. ²Shenzhen blood center, Shenzhen, P. R. China. ³Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, P. R. China.

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HIV-1 gp41 genetic diversity and enfuvirtide resistance-associated mutations among enfuvirtide-naïve patients in southern China

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