Table 3.

Summary of isotypic, neutralization, antigen reactivity, and Ig usage profiles of selected mAb hybridoma lines isolated from immunized 2F5 “complete” (V_H^+/+ × V_L^+/+) KI mice

		HIV-1 Neutralization IC50 (μg/ml)a					--Antigenic Reactivitiesb--				---------------------Ig usagec----------------------
Clone ID	Isotype	A. 92UG037.1	B. MN.3	B. SF162.LS	B. 6535.3	B. BG1168.1	MPER (2F5 epitope)	NIH-3T3 antigens	CL	Histones	VH rearrang. used	VH mutation type/aa location	VL rearrang. used	VL mutation type/aa location
V4-SE 2 CL1-1	IgG2b	0.12	<0.01	0.34	7.61	0.78	+++	++	+	+	KI 2F5 VDJ	none	KI 2F5 VJ	none
V4-SE 6 CL1-1	IgG2b	0.12	<0.01	0.45	4.81	1.04	+++	+	+	++	KI 2F5 VDJ	none	KI 2F5 VJ	none
V4-SE 8 CL1-1	IgG2b	0.14	0.01	0.54	5.6	1.1	+++	+	+	+	KI 2F5 VDJ	none	KI 2F5 VJ	none
V4-SE 361 CL1-1	IgG2b	0.21	0.03	3.04	2.75	2.37	+++	+	+	++	KI 2F5 VDJ	none	KI 2F5 VJ	none
V4-SP 1B5 CL1-1	IgG2b	0.32	0.06	1.18	17.3	1.53	+++	+	+	+	KI 2F5 VDJ	R:FWR1(T19M), CDR1(G38A)	KI 2F5 VJ	S:FWR2(P40), FWR3(E70)
V4-SE 18 CL1-2	IgG2b	0.06	<0.01	0.23	2.81	0.64	+++	+++	+	++	KI 2F5 VDJ	R:CDR3(T104I); S:FWR1(T3,T15), CDR1(G33)	KI 2F5 VJ	S:FWR1(G16), CDR1(V29), FWR3(E81)
V4-SE 372 CL1-1	IgG2b	0.14	<0.01	0.38	7.19	1.04	+++	+	++	+	KI 2F5 VDJ	none	KI 2F5 VJ	none
V4-SP 1G3 CL1-1	IgG2b	0.14	0.02	0.55	7.66	0.98	+++	+++	++	++	KI 2F5 VDJ	none	KI 2F5 VJ	none
V5-SP 3H6 CL1-1	IgG2b	0.24	0.03	2.42	2.37	2.55	+++	+	+	+	KI 2F5 VDJ	none	KI 2F5 VJ	none
V5-SE 4 CL1-1	IgG2b	0.13	<0.01	0.38	6.48	1.04	+++	+	+	+	KI 2F5 VDJ	none	KI 2F5 VJ	none
V5-SE 10 CL1-1	IgG2b	0.16	0.01	0.5	9.88	0.8	+++	+	+	+	KI 2F5 VDJ	none	KI 2F5 VJ	none
V5-SP 3B11G	IgG2c	0.13	<0.01	0.38	7.23	0.97	+++	++	++	+	KI 2F5 VDJ	none	KI 2F5 VJ	none
V4-SE 196 CL1-1	IgM	0.16	0.09	1.35	5.03	3.11	+++	+++	++	++	KI 2F5 VDJ	none	KI 2F5 VJ	none
V5-SE 14 CL1-2	IgM	0.41	0.29	5.01	22.86	7.43	+++	++	++	+++	KI 2F5 VDJ	none	KI 2F5 VJ	R:FWR2(L47P), CDR2 (S56R)
V5-SE1 CL1-1	IgM	0.64	0.28	2.86	>25	9.59	+++	+++	++	++	KI 2F5 VDJ	R:FWR1(L20V)	KI 2F5 VJ	R:CDR3(F98L)

^aIn addition to h2F5 (Table 2), m2F5 (recombinant mouse 2F5 IgG antibody) (11) was used as a neutralization control for IgG+ mAbs and had the following IC₅₀ values (averaged from two experiments): 92UG037.1=0.96, B.MN.3=0.52, SF162.LS=8.11, B.6536.3=18.6, and B.BG1168.1=9.81. Similarly, V3-1.4 (a mouse 2F5 IgM mAb) (12) was used a neutralization control for the IgM+ mAbs and had the following IC₅₀ values: 92UG037.1=0.25, B.MN.3=0.13, SF162.LS=2.35, B.6536.3=5.52, and B.BG1168.1=3.69.

^bQuantitative ELISAs to measure relative reactivities of purified mAbs to plate-bound SP62 peptide (encoding the nominal 2F5-specific MPER epitope), NIH-3T3 cytoplasmic and nuclear antigens, Cardiolipin (CL), and histones; criteria for relative positivity are described in Fig S6 and as previously described (12).

^ccDNAs from cloned hybridoma lines were amplified using a combination of either leader peptide-specific forward primers in the 2F5 KI expression cassette or degenerate forward HC or LC-specific forward primers (34), and reverse C region-specific degenerate primers.

PCR products were sequenced in both orientations, KI 2F5 V_H and V_L regions were analyzed for mutations using L-ALIGN software and endogenous LC usage was determined using the Ig-BLAST algorithm. Mutations in 2F5 V_H and V_L nucleotide sequences are represented for corresponding protein sequences as mutation type: R=replacement, S=substitution, N=Nonsense and mutation location; FRW1, FRW2, FRW3, FRW4=Framework Regions 1,2,3,4, respectively, CDR1, CDR2, CDR3=Complementarity Determining Regions 1,2, 3, respectively; information in brackets designate corresponding aa residue, number, and replacement (if applicable), with number nomenclature based on published 2F5 V_H and V_L aa sequences (9,52), and those in endogenous LCs, are annotated relative to Ig blast germline sequences.