Identification of a novel broadly HIV-1-neutralizing antibody from a CRF01_AE-infected Chinese donor

Figures & data

Fig. 1 Identification of eight monoclonal antibodies from a HIV-1 CRF01_AE-infected Chinese donor.

a Isolation of antigen-specific single B cells by flow cytometry. Single cells were sorted into a 96-well PCR plate containing lysis buffer according to the presented gating strategy. b Amino acid sequence analysis of monoclonal antibodies with alignment to respective germline genes. The framework region (FR) and complementarity determining region (CDR) were determined based on the program IMGT/V-QUEST. The symbol “.” denotes conserved amino acids

Gene family analysis of monoclonal antibodies

	Heavy chain					Light chain
	IGHV	IGHJ	IGHD	CDR3 (aa)	SHM (%)	IGKV	IGKJ	CDR3 (aa)	SHM (%)
F2	1–69*01	3*01	3–22*01	17	18.06	4–1*01	3*01	9	9.93
H6	1–69*01	3*01	3–22*01	17	17.01	4–1*01	3*01	9	11.70
BF8	1–69*01	3*01	3–22*01	17	14.24	4–1*01	3*01	9	10.64
F4	1–69*11	3*01	3–22*01	17	16.32	4–1*01	3*01	9	8.87
F8	1–69*11	3*01	3–22*01	17	14.58	4–1*01	3*01	9	7.80
BE7	1–69*11	3*01	3–22*01	17	16.32	4–1*01	3*01	9	7.80
F6	4–34*01	6*01	2–8*01	18	16.14	2–28*01	2*02	9	11.83
BE10	3–23*04	6*02	3–16*02	21	19.44	1–16*01	1*01	9	10.61

The program IMGT/V-QUEST was applied to analyze the gene germline, complementarity determining region (CDR) 3 length, and somatic hypermutation (SHM). The SHM frequency was calculated from the mutated nucleotides

Fig. 2 Purification and identification of monoclonal binding antibodies.

a Reducing 12% SDS-PAGE analysis of monoclonal antibodies. b ELISA binding of monoclonal antibodies with the sorting probe BG505. c ELISA of cross-reactive monoclonal antibodies binding with diverse HIV-1 GP140 antigens including clade B, CRF01_AE, and CRF07_BC

Neutralizing activity of monoclonal antibodies on the Global Panel and two Tier 1 viruses from the DRVI Panel

				Global panel
	Pseudovirus	SF162	MW965	398F1	TRO11	X2278	25710	CE0217	CE1176	X1632	CNE55	CNE8	BJOX2000	CH119	246F3
	Clade	B	C	A	B	B	C	C	C	G	CRF01_AE	CRF01_AE	CRF07_BC	CRF07_BC	AC
	Tier	1	1	2	2	2	2	2	2	2	2	2	2	2	2
F2		0.06	0.38	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
H6		0.34	0.99	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
BF8		0.04	0.12	23.98	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
F4		0.04	0.02	49.42	>50	>50	38.26	>50	>50	>50	>50	>50	>50	>50	>50
F8		0.01	0.18	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
BE7		0.02	0.04	>50	>50	>50	44.19	>50	>50	>50	>50	>50	>50	>50	>50
F6		>50	>50	10.00	>50	31.94	46.00	>50	13.53	8.21	41.07	34.85	>50	>50	8.27
BE10		>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50	>50
VRC01		0.48	0.07	0.41	0.77	0.35	0.87	0.42	4.88	0.31	0.64	0.56	>50	1.56	0.51

Neutralizing potency was measured as IC₅₀ in μg/ml of the monoclonal antibodies. Values <0.2 μg/ml in bold, 0.2–2 μg/ml in italic, and 2–50 μg/ml in underline

Neutralizing activity of F6 against diverse HIV-1 isolates on the DRVI Panel

	Pseudovirus	Q461	Q769	Q259	Q842	SF162	QH0692	SC422661	PVO.4	AC10	RHPA4259	REJO4541	TRJO4551	CAAN5342
	Clade	A	A	A	A	B	B	B	B	B	B	B	B	B
	Tier	2	2	2	2	1	2	2	3	2	2	2	3	2
F6		>50	3.89	>50	>50	>50	21.78	25.57	>50	17.29	19.28	>50	2.58	31.78
VRC01		0.65	0.12	0.18	0.09	0.48	1.72	0.51	1.09	3.51	0.12	0.34	0.41	1.72
	Pseudovirus	MW965	ZM109	Du422	ZM249M	CAP45	CH110	CH117	CH181	CH120	BJMSM2249	BJMSM2316	BJMSM2498
	Clade	C	C	C	C	C	CRF07_BC	CRF07_BC	CRF07_BC	CRF07_BC	CRF01_AE	CRF01_AE	CRF01_AE
	Tier	1	1	2	2	2	2	2	2	3	ND	ND	ND
F6		>50	>50	18.97	>50	>50	17.82	>50	>50	43.56	2.50	0.91	1.56
VRC01		0.07	0.52	>50	0.24	9.09	1.34	0.36	1.14	6.90	3.77	0.32	1.50

ND not determined

Neutralizing potency was measured as IC₅₀ in μg/ml of the monoclonal antibodies. Values <0.2 μg/ml in bold, 0.2–2 μg/ml in italic, and 2–50 μg/ml in underline

Fig. 3 The newly isolated F6 recognizes the linear epitope on GP120.

a ELISA binding of F6 to HIV-1 GP140 and GP120 antigens. b–d ELISA binding of F6 to native and reduced BG505 GP140, CN54 GP140, and CN54 GP120 treated with 10 mM DTT at 37 °C for 1 h. e–g ELISA binding of F6 to native and denatured BG505 GP140, CN54 GP140, and CN54 GP120 treated with denaturing buffer (New England Biolabs) at 100 °C for 10 min

Fig. 4 F6 does not target the CD4 binding site epitope of GP120.

a ELISA binding of F6 to RSC3 and ΔRSC3. b ELISA binding of F6 to TriMut and TriMut/368/370/474. c Competition neutralizing of F6 against HIV-1_BJMSM2316 with TriMut and TriMut/368/370/474. d Flow cytometry dot plots showing the binding capacity of F6 to HIV-1_BJMSM2316 and HIV-1_{BJMSM2316_D368R} GP160 on the surface of transfected 293 T cells. e The percentage of FITC + cells in 293 T cells are shown as mean ± SD (n = 3). The symbol “**” indicates P < 0.01

Fig. 5 Alternative epitope identification of F6.

a Neutralization of F6 against HIV-2_7312A and HIV-2_7312A-C1. b Neutralization of F6 against HIV-1_BG505 and HIV-1_{BG505_T332N}. c Neutralization of F6 against HIV-1_BJMSM2316 and HIV-1_{BJMSM2316_N332A}. d Neutralization of F6 against HIV-1_BJMSM2316 and HIV-1_{BJMSM2316_N160K}. e–g ELISA binding of F6 to deglycosylated CN54 GP140 and GP120 treated with Endo H enzyme (New England Biolabs) at 37 °C according to the manufacturer’s protocol