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. Author manuscript; available in PMC: 2014 May 10.
Published in final edited form as: Virology. 2013 Mar 5;439(2):81–88. doi: 10.1016/j.virol.2012.12.010

Cross-neutralizing activity of human anti-V3 monoclonal antibodies derived from non-B clade HIV-1 infected individuals

Raiees Andrabi a,c, Constance Williams a, Xiao-Hong Wang b, Liuzhe Li a, Alok K Choudhary c, Naveet Wig c, Ashutosh Biswas c, Kalpana Luthra c, Arthur Nadas d, Michael S Seaman e, Phillipe Nyambi a,b, Susan Zolla-Pazner a,b, Miroslaw K Gorny a,*
PMCID: PMC3756680  NIHMSID: NIHMS451797  PMID: 23466102

Abstract

One approach to the development of an HIV vaccine is to design a protein template which can present gp120 epitopes inducing cross-neutralizing antibodies. To select a V3 sequence for immunogen design, we compared the neutralizing activities of 18 anti-V3 monoclonal antibodies (mAbs) derived from Cameroonian and Indian individuals infected with clade AG and C, respectively. It was found that V3 mAbs from the Cameroonian patients were significantly more cross-neutralizing than those from India. Interestingly, superior neutralizing activity of Cameroonian mAbs was also observed among the nine VH5-51/VL lambda genes encoding V3 mAbs which mediate a similar mode of recognition. This correlated with higher relative binding affinity to a variety of gp120s and increased mutation rates in V3 mAbs from Cameroon. These results suggest that clade C V3 is probably weakly immunogenic and that the V3 sequence of CRF02_AG viruses can serve as a plausible template for vaccine immunogen design.

Keywords: HIV-1, V3 region, Non-B clade HIV-1, Immunoglobulin gene usage, Human monoclonal antibodies, HIV neutralizing antibodies

Introduction

In light of the continuing worldwide spread of HIV-1 infection, it is accepted that a vaccine would be the most effective prophylaxis, although the genetic variation of the virus remains a major challenge. A certain optimism for designing an efficient vaccine was recently raised by the results from the RV144 clinical vaccine trial, which suggested that anti-HIV-1 antibodies (Abs) played a protective role (Rerks-Ngarm et al., 2009). Indeed, it was revealed that high titers of plasma anti-V1/V2 Abs correlated inversely with the rate of HIV-1 infection (Haynes et al., 2012). The neutralizing antibody response in the vaccine recipients was weak and did not correlate with the rate of infection but it was suggested that it can be partially protective against HIV-1 (Montefiori et al., 2012).

One concept for an HIV vaccine is to design a template with a set of immunogens representing various epitopes in the envelope. Such epitopes could include the V1/V2 regions as well as the V3 loop which is known to induce cross-clade neutralizing Abs (Corti et al., 2010; Hioe et al., 2010; Pantophlet et al., 2007; Scheid et al., 2009;2011). The contribution of anti-V3 Abs in protection against HIV-1 infection was documented by passive immunization experiments in animal models (Andrus et al., 1998; Emini et al., 1992; Watkins et al., 2011). Studies in vitro in several laboratories showed that the human V3 mAbs can neutralize the majority of tier 1 pseudoviruses (psVs) and some tier 2 viruses but their cross-clade neutralizing activity is relatively limited (Corti et al., 2010; Hioe et al., 2010; Pantophlet et al., 2007; Scheid et al., 2009;2011). Moreover, it was demonstrated in animal experiments that V3 immunogens can induce cross-clade neutralizing antibodies (Letvin et al., 2001; Zolla-Pazner et al., 2011).

The question still remains which V3 structure would be the most effective in inducing cross-neutralizing Abs. It was shown previously that anti-V3 mAbs derived from individuals infected with non-B clade viruses were more cross-reactive than clade B-derived V3 mAbs (Gorny et al., 2006). This appears to be due to the fact that the relatively conserved V3 motif GPGR in clade B viruses focuses the immune response on the positively charged Arg (R) and thus narrows the cross-reactivity of the responding V3 Abs, while the GPGQ motif in non-B clade viruses allows induction of Abs to other conserved elements of the V3 region (Jiang et al., 2010). The non-B clade viruses, with the GPGQ motif, are a very heterogeneous group, involved in ~85% of global HIV-1 infection with the most dominant being clade C, followed by clade A (Osmanov et al., 2002).

In this study we compared the neutralizing and binding activity of two panels of anti-V3 mAbs derived from subjects living in Cameroon and India and infected with non-B clade viruses containing the envelope (Env) sequence corresponding to clade A, mainly CRF02_AG, and clade C, respectively. The results showed that the mAbs from the Cameroonian patients were significantly more cross-neutralizing than those from India, suggesting that the V3 structure and sequence of CRF02_AG viruses would be better for immunogen design.

Results

Human anti-V3 mAbs

A panel of 18 anti-V3 mAbs derived from individuals infected with non-B clade HIV-1 includes five new anti-V3 mAbs, not yet described, generated from Cameroonian (mAbs 4210 and 4508) and Indian patients (mAbs 4139, 4487 and 4490) (Table 1). These five new mAbs belong to subclass IgG1 with four λ and one κ light chains (Table 1). The remaining 13 V3 mAbs have been previously described and characterized (Table 1).

Table 1.

Human anti-V3 and control mAbs used for the study.

No. mAbsa Site Isotype Subtype of the infecting virusb Country of origin Reference
1 3869 V3 IgG1 λ Nd Cameroon Gorny et al. (2009)
2 4210 V3 IgG1 λ Nd Cameroon This study
3 3074 V3 IgG1 λ CRF02_AG Cameroon Gorny et al. (2006)
4 3881 V3 IgG1 κ CRF02_AG Cameroon Gorny et al. (2009)
5 4508 V3 IgG1 κ CRF02_AG Cameroon This study
6 2557 V3 IgG1 λ CRF02_AG Cameroon Gorny et al. (2004)
7 2558 V3 IgG1 λ CRF02_AG Cameroon Gorny et al. (2004)
8 3019 V3 IgG1 λ CRF02_AG Cameroon Gorny et al. (2006)
9 3694 V3 IgG1 λ H Cameroon Gorny et al. (2009)
10 4139 V3 IgG1 λ C India This study
11 3791 V3 IgG1 κ C India Gorny et al. (2009)
12 4121 V3 IgG1 λ C India Gorny et al. (2009)
13 3904 V3 IgG1 κ C India Gorny et al. (2009)
14 3906 V3 IgG1 λ C India Gorny et al. (2009)
15 4490 V3 IgG1 λ Nd India This study
16 4022 V3 IgG1 λ C India Gorny et al. (2009)
17 4025 V3 IgG1 λ Nd India Gorny et al. (2009)
18 4487 V3 IgG1 λ Nd India This study
Controls: 447-52D V3 IgG3 λ B USA Gorny et al. (1993)
1418c B19 IgG1 κ N/A USA Gigler et al. (1999)
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Nd—Not determined.

a

Each mAb is derived from one HIV-1 infected individual (except 1418 which was produced from a subject carrying anti-parvovirus B19 serum Abs) living in the depicted countries.

b

The subtype of the infecting HIV-1 was determined in our laboratory by sequencing the envelope fragment (C2-V5) from the donor plasma virus.

c

Human mAb 1418 is specific against parvovirus B19 and it serves here as negative control.

Immunoglobulin gene usage

The variable fragment of the heavy (VH) and light (VL) chain genes were sequenced and analyzed using the IMGT system to determine the immunoglobulin (Ig) gene usage and percentage of mutations. A biased usage of VH genes was observed, as 9 of 18 (50%) anti-V3 mAbs were encoded by the VH5-51 gene segment: four mAbs developed from Cameroonian and five from Indian patients; however, these mAbs used different alleles, mainly *03 and *01, respectively (Table 2). Five other mAbs used VH1 family genes while three mAbs, 3074, 3881 and 4508 used one particular gene segment, VH4-59. The VH3 family genes, which are the most commonly used by Abs derived from healthy individuals (Tiller et al., 2007), were represented only by one V3 mAb which used the VH3-30 gene (Table 2).

Table 2.

Analysis of immunoglobulin gene usage and percent mutations in the variable fragment of human anti-V3 mAbs.

mAba IGHVb IGHJb mut (%)c IGLVb IGLJb mut (%)c
3869 1-f*01 4*02 8.3 L3-1*01 2*01/3*01 8.6
4210 1-f*01 3*02 5.6 L3-1*01 3*02 4.3
3074 4–59*01 3*01 5.9 L1-51*02 2*01/3*01 5.3
3881 4–59*01 3*02 11.2 K3-15*01 1*01 5.4
4508 4–59*01 4*02 4.6 K4-1*01 3*01 4.7
2557 5–51*01 3*01 14.2 L3-1*01 3*02 14.0
2558 5–51*03 4*02 7.3 L3-1*01 2*01/3*01 5.7
3019 5–51*03 3*01 5.9 L3-1*01 2*01/3*01 9.7
3694 5–51*03 1*01 18.1 L3-1*01 2*01/3*01 11.5
4139 1–8*01 4*02 6.6 L2-14*01 2*01/3*01 6.6
3791 1–18*01 4*02 12.2 K1-3301 4*01 4.3
4121 1–18*01 4*02 5.2 L1-44*01 1*01 1.4
3904 3–30*03 4*03 5.9 K1-39*01 3*01 5.0
3906 5–51*01 3*02 7.3 L3-1*01 2*01/3*01 9.7
4490 5–51*01 4*02 4.5 L3-1*01 2*01/3*01 5.0
4022 5–51*01 5*02 11.8 L3-10*01 3*02 7.9
4025 5–51*01 6*02 3.8 L3-10*01 1*01 4.7
4487 5–51*03 4*02 8.3 L1-51*02 2*01/3*01 5.6
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a

The first nine mAbs are derived from Cameroon while the second nine mAbs are from India HIV-1 infected subjects, separated by a space.

b

Immunoglobulin genes and alleles (*) were determined using the nucleotide sequence of the variable fragments of mAbs analyzed by IMGT system.

c

mut — percent of mutation in IGHV and IGLV indicate the difference in nucleotide sequence between the tested mAbs and corresponding germline.

Usage of the light chain genes was also biased toward lambda genes which were used by 14 of 18 mAbs (Table 2) while in mAbs derived from healthy subjects there is dominance of κ over λ light chain genes (Tiller et al., 2007). Among lambda VL genes, the most frequently used was the VL3-1 gene in 8 of 14 mAbs (57%), which mainly paired with VH5-51 gene (6 of 9 pairs using VH5-51 gene).

The amino acid sequence of the complementarity-determining region 3 (CDR) of the heavy chain (H3) and light chain (L3) was unique for each mAb (Table S1).

Neutralization of pseudoviruses by anti-V3 mAbs derived from Cameroonian and Indian HIV-1 infected patients

A panel of 18 anti-V3 antibodies derived from the Cameroonian and Indian HIV-1 infected subjects and a control mAb 1418 (anti-parvovirus B19) were tested with 41 pseudotyped viruses from clade A, B, C and AG for their neutralizing activity. Twenty-one of 41 viruses tested were found to be neutralized by this panel of V3 mAbs with a 50% inhibitory concentration (IC50) < 50 μg/ml (Table 3). The remaining 20 psVs were not neutralized at an IC50 below 50 μg/ml (data not shown); all but one of these were tier 2 viruses (HO29.12, HO30.7, HO35.18, HO61.14, WITO4160.33, SC42661.8, TRO.11, AC10.0.29, THRO4156.18, CAAN5342.A2, PVO.4, TRJO4551.58 [clade B], CAP45.2.00.G3, Du156.12, Du172.17, Du422.1, ZM53M.PB12, ZM135M.PL10, ZM214M.PL15, ZM249M.PL1 [clade C]).

Table 3.

Neutralization of pseudoviruses by anti-V3 mAbs derived from Cameroonian and Indian HIV-1 infected individualsa.

No. Virus ID Tier Clade Cameroon mAbs
India mAbs
2558 2557 3019 3694 3869 4210 3074 4508 3881 3906 4022 4487 4490 4025 3791 4121 4139 3904 1418
5-51 5-51 5-51 5-51 1-f 1-f 4-59 4-59 4-59 5-51 5-51 5-51 5-51 5-51 1-18 1-18 1-8 3-30
1 MW965.26 1A C <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 –
2 271-11 1B AG 2 3.6 5.5 19.5 0.9 1.2 <0.4 <0.4 – 0.4 3.2 3.1 – 3.9 <0.4 17.9 1.3 3.4 –
3 DJ263.8 1B A 1.9 – – 4.4 2.2 – <0.4 <0.4 <0.4 3.1 0.7 – 9.1 0.8 <0.4 6.2 0.6 1.7 –
4 25710-2.43 1B C – – – – – – 11.7 14.5 46 – – – – – 43 – – – –
5 242-14 1B AG – – – – – – 49.9 – – – – – – – – – – – –
6 SF162.LS 1A B <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 <0.4 – – 18.4 <0.4 3.7 3.2 <0.4 – 12 25.8 – –
7 Bx08.16 1B B 0.8 0.4 0.8 6.9 1.1 3.9 3.3 – – – 10.6 2.8 2.8 – – 32.3 23.7 – –
8 BaL.26 1B B 0.4 <0.4 0.7 1.4 1.4 3.7 5.2 – – – – 4.7 4 – – 37.8 – – –
9 SS1196.1 1B B 2.6 2.6 5.2 25.5 1 10.7 7.5 – – – 7.8 14.4 21.8 – – – – – –
10 BZ167.12 1B B 0.5 – – 2.5 – 30.2 – – – 0.4 – – – – – 4 – – –
11 HXB2.DG 1B B 45.4 – – – – – – – – 9.4 – – – – – – – – –
12 ZM109F.PB4 2 C 24.9 25.3 – – 14.5 15.7 6.6 2.5 – 5.1 26.6 – – 49.1 8.7 – – – –
13 ZM233M.PB6 2 C – – – – 44.3 – 49.7 – – 37.8 – – – – – – – – –
14 ZM197M.PB7 2 C – – – – – – – – – – – 16.7 – – – – – – –
15 CAP210.2.00 2 C – – – – – – 39.5 – – – – – – – – – – – –
16 6535.3 2 B 32.6 2.9 – – 1.6 – – – – – – – – – – – – – –
17 QH0692.42 2 B 46.1 41.8 – – 9.9 – – – – – – – – – – – – – –
18 HO31.7 2 B 13.9 5.9 13.2 – – – – – – – – – – – – – – – –
19 JRFL.JB 2 B 30.4 21.2 47.5 – – – – – – – – – – – – – – – –
20 REJO4541.67 2 B – 14.1 – – 49.6 – – – – – – – – – – – – – –
21 RHPA4259.7 2 B – – – – 26.1 – – – – – – – – – – – – – –
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a

All 18 mAbs were tested against 41 pseudotyped viruses using the TZM-bl cell assay; only 21 viruses, reactive with at least one mAb are shown in the table. Nine mAbs from each group (Cameroon and India) are shown with sample IDs (4 digit numerical) at the top of each column and immunoglobulin VH gene usage in the next row below. Monoclonal Abs encoded by VH5-51 gene are bold. The space separates tier 1 from tier 2 pseudotyped viruses. The IC50 values which indicate the amount of mAbs (mg/ml) needed for 50% neutralization are shown in each cell; they were estimated from the titration curves. The IC50 >50 ug/ml (–) have been omitted. Monoclonal Ab 1418 is specific to parvovirus B19 and is used here as a negative control.

The V3 mAbs neutralized both sensitive tier 1 and the more resistant tier 2 psVs; however, the mAbs displayed different patterns of activity with these two categories of psVs. For example, most tier 1 viruses were neutralized at < 1 μg/ml, while most of the tier 2 viruses required > 10 μg/ml of the mAbs to reach 50% neutralization. In terms of frequency, 106 of 198 (53%) tier 1 psVs/mAb combinations showed neutralizing activity while only 30 of 180 (16%) tier 2 psVs/mAb combinations showed neutralization (p < 0.001) (Table 3).

Interestingly, nine mAbs from the Cameroonian patients neutralized 21 psVs significantly more potently than the nine mAbs from the Indian patients by comparing their IC50 values (p=0.001). The significantly more potent neutralizing activity of the anti-V3 mAbs from the Cameroonian patients was observed separately for (a) tier 1 (n=11 psVs, p < 0.01), (b) tier 2 (n=10 psVs, p < 0.0001), (c) clade B (n=12 psVs, p=0.002), and (d) non-B clade viruses (n=9 psVs, p=0.01). Statistical significance was determined using the Chi-Squared test.

In terms of frequency, which also reflects cross-neutralization, 83 of 189 psVs/mAb combinations (44%) of the mAbs from the Cameroonian patients displayed neutralization compared to 53 of 189 psVs/mAb combinations (28%) displayed by the mAbs from the Indian patients (Table 3).

To understand the difference in the neutralizing activity we analyzed (a) Ig gene usage, (b) percentage of mutations, (c) relative binding affinity and (d) the epitopes of the mAbs derived from the Cameroonian and Indian patients.

Neutralization of VH5-51- and non-VH5-51-derived anti-V3 mAbs

Nine of 18 V3 mAbs, including four from Cameroonian and five from Indian patients, used the VH5-51 gene paired with VL lambda genes; six VL3-1, two VL3-10 and one VL1-51 gene (Table 2). Using the neutralization data from Table 3, statistical analysis revealed that the VH5-51-derived V3 mAbs from the Cameroonian patients were significantly more potent than the corresponding mAbs from the Indian patients (p=0.006, Table S2). Similarly, we analyzed the remaining mAbs encoded by non-VH5-51 genes including the VH1 family, the VH4-59 and VH3-30 genes. It was also found that the mAbs from the Cameroonian compared to Indian patients were significantly more potent in neutralization of psVs (p=0.01, Table S3) as determined using the Chi-Squared test, suggesting that the neutralizing efficiency of V3 mAbs from the Cameroonian patients may be independent of their VH gene usage.

Percentage of mutations in the heavy and light chain genes of anti-V3 mAbs

The percentage of point mutations in the nucleotide sequences of the variable (V) region of the heavy and light chain genes was determined by alignment with the corresponding germline genes using the IMGT system. The percentage of mutations varied for the VH genes from 3.8% to 18.1%, with a mean of 8.1%, while for VL genes varied from 1.4% to 14.0% with a mean of 6.6% (Table 2). We compared the percentage of mutations in the heavy and light chain genes combined together using the Student’s t-test; the mean percentage for mAbs derived from the Cameroonian patients was higher than for those from the Indian patients, 16.7% versus 12.8%, but the difference was not significant (p=0.20) (Table S4).

The percentage of mutations was also analyzed separately for VH5-51-encoded V3 mAbs and was higher (although not significantly) for mAbs derived from the Cameroonian patients compared to those from the Indian patients, 21.6% versus 13.7% (p=0.13), respectively. The non-VH5-51 mAbs, which used the VH1 family, VH4-59 and VH3-30 genes, had only a slightly higher percentage of mutations amongst the mAbs from Cameroon vs. those from India, 12.7% and 11.8% (p=0.71) (Table S4). These results indicate that the percentage of mutations is higher among the V3 mAbs from Cameroon compared to those from India, especially in VH5-51-derived mAbs, but statistically comparable.

We analyzed the possible association between the percentage of mutations in the 18 V3 mAbs and the number of neutralized pseudoviruses and we found only a trend but not a significant correlation (p=0.2668 for all psVs and p=0.1927 for tier 2 psVs; Fig. S1). This study has some limitations because the CDR3 sequence of the heavy chain (CDR H3) is not included in the analysis of mutation rate due to lack of the corresponding germline sequence.

Binding of anti-V3 mAbs to recombinant gp120s

The lower neutralizing activity of the V3 mAbs from the Indian patients compared to those from the Cameroonian patients could be related to lower binding affinity. To test this hypothesis, all 18 anti-V3 mAbs were tested for their reactivity to 18 recombinant gp120s with sequences from one clade A, seven clade C and 10 clade B viruses (Table 4). Twelve gp120s corresponded to the respective psVs tested in the neutralization assay, but only four of these psVs (SF162.LS, ZM197M.PB7, JRFL.JB and REJO4541.67) were neutralized, as shown in Table 3. Although recombinant gp120 cannot mimic the native trimeric structure of the envelope which is targeted by the mAb in order to neutralize, binding indicates the presence of the epitope in the virus envelope. A representative binding pattern of mAbs to gp120s of clade C, A and B supported these data (Fig. S2A–D).

Table 4.

Relative affinity binding of mAbs to recombinant gp120sa.

gp120 Clade Cameroon mAbs
India mAbs
2557 2558 3694 3019 3869 4210 3074 3881 4508 4022 4490 4487 3906 4025 4121 4139 3791 3904
5-51 5-51 5-51 5-51 1-f 1-f 4-59 4-59 4-59 5-51 5-51 5-51 5-51 5-51 1-18 1-8 1-18 3-30 pb
Du151 C 0.026 0.010 0.042 0.020 0.013 0.064 0.028 0.010 0.024 0.003 0.012 0.024 0.025 0.010 0.075 0.045 0.024 0.020
Du156.12 C 0.010 0.005 0.005 0.001 0.027 0.025 0.019 0.005 0.011 0.002 0.010 0.019 0.017 0.008 0.009 0.027 0.013 0.002
Du422.1 C 0.006 0.003 0.007 0.002 0.019 0.010 0.018 0.004 0.006 0.001 0.007 0.017 0.008 0.006 0.011 0.022 0.007 0.003
ZM53M.PB12 C 0.006 0.006 0.003 0.006 0.008 0.009 0.013 0.002 0.006 0.001 0.008 0.011 0.012 0.004 0.006 0.007 0.002 0.002 0.529
Du172.17 C 0.017 0.009 0.018 0.001 0.024 0.021 0.024 0.009 0.017 0.003 0.021 0.028 0.021 0.014 0.045 0.024 0.009 0.007
C.ZA.1197MB C 0.009 0.004 0.007 0.001 0.020 0.014 0.015 0.004 0.015 0.001 0.014 0.016 0.068 0.005 0.018 0.023 0.009 0.002
ZM197M.PB7 C 0.017 0.006 0.007 0.005 0.019 0.012 0.017 0.004 0.030 0.003 0.038 0.030 0.012 0.021 0.040 0.027 0.003 0.005
92 RW020 A 0.030 0.016 0.006 0.001 0.033 0.029 0.033 0.008 0.021 0.002 0.008 0.018 0.029 0.005 0.009 0.023 0.010 0.003
SF162 B 0.058 0.011 0.009 0.004 0.031 0.025 0.019 0.160 – 0.001 0.008 0.011 0.020 0.004 0.014 0.023 – –
WITO4160 B 0.027 0.004 0.012 0.007 0.008 0.018 0.017 0.434 – 0.006 0.014 1.934 0.198 0.798 0.050 0.020 – –
TRO B 0.077 0.010 0.021 0.022 0.006 0.029 0.022 0.949 – 0.074 0.046 – – – 0.097 0.096 – –
JRFL B 0.018 0.008 0.008 0.002 0.019 0.016 0.072 – – 0.053 0.027 0.029 – – 0.174 – – –
PVO B 0.019 0.007 0.018 0.008 0.008 0.024 0.094 – – 0.003 0.032 0.032 0.023 0.008 – 0.072 – – 0.0002
REJO4541 B 0.020 0.009 0.026 0.009 0.009 0.631 0.022 – – 0.004 0.029 1.697 – 0.074 – – – –
SC422661.8 B 0.022 0.011 0.015 0.010 0.010 0.016 1.473 – – 0.024 0.009 0.013 0.024 0.076 – – – –
MN B 0.055 0.026 0.026 0.005 0.029 0.037 – – – 0.016 0.027 0.023 0.090 – 0.122 – – –
CDC451 B 0.011 0.259 0.008 – 0.050 – – – – – – – – – – – – –
11.B11.1550 B 0.137 0.296 – – – – – – – – – – – – – – – –
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a

Nine mAbs from each group (Cameroon and India) were tested by ELISA against 18 recombinant gp120s coated onto plastic plate at 1 μg/ml. All mAbs were titrated at a concentration ranging from 10 to 0.00001 μg/ml (12 dilutions). At the top of each column the ID of the mAb is shown and immunoglobulin gene VH usage in the next row below; VH5-51-derived mAbs from each panel are bold. The space separates gp120 clade C and A from clade B. The binding activity of V3 mAbs to gp120 is shown as 50% of maximal binding and represents the relative affinity. Human anti-parvovirus B19 mAb 1418 was used as a negative control and was not reactive (data not shown). Each experiment was performed at least two independent times. —no reactivity (> 10.0).

b

p — p Value was determined using the Chi-Squared longrank test; it indicates significantly higher relative affinities of Cameroonian compared to Indian mAbs for binding to clade B gp120s.

The binding experiments showed that the anti-V3 mAbs were able to bind all the clade C and one clade A gp120s and the only difference between the mAbs from Cameroon and India was observed in binding to clade B gp120s (Table 4). Particularly, none of the mAbs from the Indian panel was able to bind two of the clade B gp120s (CDC451 and 11.B11.1550) while four mAbs from the Cameroonian panel showed reactivity (Table 4, Fig. S2D).

The relative affinities measured by 50% of maximal binding were higher for Cameroonian versus Indian V3 mAbs; for binding to clade C and A gp120s the difference was not significant (p=0.529) while for binding to clade B gp120s the difference was highly significant (p=0.0002, Chi-Squared test) (Table 4).

Study of the V3 epitopes based on mAbs binding to gp120s

The binding experiments revealed that clade C and clade A gp120s were recognized by all V3 mAbs in contrast to clade B gp120s that was reactive with majority of mAbs. Three mAbs, 4508, 3791 and 3904 (derived from one Cameroonian and from two Indian patients) did not bind to any of the clade B gp120s which differ from clade C and A gp120s by residue Arg (R) at position 315 in the V3 region (Tables 4 and 5) suggesting that the epitope of these mAbs include the tip of the V3 loop. The remaining 15 mAbs were tolerant for changes in R/Q/K residues at position 315, suggesting that this residue is not an important component of the epitope. Furthermore, the mAbs were sensitive to the presence of two conserved residues, I309 and F317, suggesting that these two highly conserved residues on the hydrophobic face of V3 (Gorny et al., 2011; Jiang et al., 2010) are involved in the epitopes of 15 mAbs. This conclusion is supported by the fact that mutations at these two positions, in the gp120s of CDC451 and 11.B11.1550 (Table 5) abrogated or substantially reduced the binding of the majority of the mAbs (Table 4).

Table 5.

V3 sequences of recombinant gp120s tested in the binding assay.

gp120 Clade V3 sequencea
Consensus C CTRPNNNTRKSIRIGPGQTFYATGDIIGDIRQAHC
296 315 323a 331
Du156.12 C –––––––––––V–––––––––––––––––––––––
Du172.17 C ––––S––––––V––––––––F––––––––––––––
Du422.1 C –––––––––––V––––––––––––E––––––E–––
Du151 C ––––––––––––––––––––––––E–––N––E–––
ZM53M.PB12 C ––––G–––––––––––––A–F––TN––––––––Y–
C.ZA.1197MB C –––––––––––––––––––A––––NE––––––––Y–
ZM197M.PB7 C –V–––––––––V––––––––F–––E–––––––––––
92 RW020 A ––––––––––GV––––––A–––––G––––––––––
PVO B ––––––––––––S––––RA––––––––––––––––
SF162 B ––––––––––––T––––RA––––––––––––––––
TRO B –––––––––R––H––––RA––––––––––––––––
REJO4541 B ––––––––––––H–A––RA–––––E––––––K–Y–
WITO4160 B ––––G––––R––N––––RA–––––A––––––K–––
MN B –––––Y–K––R–H––––RA––T–KN–K–T––––––
SC422661.8 B ––––––––––G–T––––RV––*––E–V–––––V––
JRFL B ––––––––––––H––––RA––T––E––––––––––
CDC451 B ––––––H–––RVTL–––RVW–T––E–L–N––––––
11.B11.1550 B –I––––––––R–TM–––KVY–T––Q––––––––––
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a

The V3 loop sequences of gp120s were retrieved from the Los Alamos HIV-1 database and were aligned with consensus C V3 sequence (shown at the top) using the seqpublish program (http://www.hiv.lanl.gov). The numbering of the V3 consensus C sequence corresponds to HXB2 gp160 position numbers 296-331 with a two residue deletion (310=Q and 311=R) and one residue insertion (323a=I); this was determined using the HIV Sequence Locator program (http://www.hiv.lanl.gov). To emphasize the numbering of V3 consensus C, the four residues are underlined and numbered below: two cysteines (C) at positions 296 and 331, a glutamine (Q) at 315 and isoleucine (I) at 323a. The V3 crown is comprised of approximately 13 amino acids (bold), to which all the mAbs bind. The residues Arg (R) and Lys (K) both at position 315 are present in the clade B viruses and are bold and underlined. The hydrophobic residues I309 and F317 are prerequisite for binding most of the mAbs tested; mutations in these positions, bold and underlined, result in limited reactivity of gp120s with V3 mAbs.

Discussion

Analysis of human mAbs that display cross-neutralizing activity to diverse HIV-1 subtypes is critical for gaining insight into the design of a prophylactic vaccine. We previously showed that non-B clade anti-V3 mAbs are more potent and cross-neutralizing than clade B derived mAbs (Gorny et al., 2006). Notably, in the present study we found that the neutralizing activity of mAbs derived from non-B clade-infected individuals can be significantly different depending on whether the donor was infected by strains circulating in Cameroon, mainly CRF02_AG or with strains circulating in India, mainly clade C HIV-1. The greater neutralizing activity of the Cameroon-derived V3 mAbs was unexpected given the close resemblance of the consensus V3 sequences between CRF02_AG and clade C viruses (only one residue Ile is replaced by Val in the entire V3 sequence; …RKSI/VRIGPGQ…) and the conservation of the GPGQ motif in the V3 crown of both clades.

Similarly, the Cameroon-derived mAbs were more potent than the Indian-derived mAbs even when the mAbs were encoded by the same VH5-51 and VL lambda genes. We have already observed that human anti-V3 mAbs display biased usage of Ig genes, preferentially using the pair of VH5-51 and VL lambda genes which is most likely related to an antigen requirement (Gorny et al., 2011;2009). The superior neutralizing activities of Cameroonian versus Indian V3 mAbs coded for by VH5-51/VL lambda genes suggest that this is related to higher binding affinity rather than different epitope recognition since these mAbs have similar shape of the antigen-binding site and bind the same V3 epitopes (Gorny et al., 2011; Jiang et al., 2010). The binding studies confirmed that relative affinities of Cameroonian compared to Indian V3 mAbs were significantly higher for binding to clade B gp120s and comparable with certain trends for higher binding to clade C and A gp120s. Thus the mAbs from India possibly did not mature as efficiently, resulting in their lower affinity and lower neutralizing activities. Lastly, it cannot be excluded that host genetics contributed differently in regulating the virus-specific antibody response in the two study populations.

Studies of the mutation rates are consistent with this hypothesis, as the antibodies from India compared to those from Cameroon had a lower percentage of mutations in the variable fragment of the heavy and light chain genes, particularly in mAbs encoded by the same VH5-51/VL lambda genes (though the difference did not reach statistical significance). Thus, it has been suggested that the percentage of mutations in VH and VL may reflect affinity maturation and length of antigenic stimulation. For example, it was observed that human anti-gp120 mAbs produced from B cells derived from recipients of a gp120 HIV-1 vaccine had 3.8% (range 0 to 8.2%) of V(H) somatic mutations after six months of immunization (Moody et al., 2012). In contrast, broadly neutralizing mAbs, which usually appear after 2–3 years of HIV-1 infection, have 20%–35% of mutations (Walker et al., 2011;2009; Wu et al., 2010). Another explanation may be the greater range of V3 sequences that are found in AG-infected individuals compared to clade C-infected individuals in which the V3 region is quite constant (Patel et al., 2008). Thus, antibody maturation could reflect both length of antigenic stimulation and the diversity of the antigen driving the immune response.

Given the similar recognition mode of the majority of the mAbs described here, the question arose why mAbs from India, even using the same VH5-51/VL lambda genes, have significantly lower neutralizing potency and relative affinity compared to those from Cameroon. The data suggest that V3 of clade C compared to clade AG viruses have lower immunogenicity resulting from different V3 structures which impact the maturation of anti-V3 antibodies.

This hypothesis is supported by two studies that analyzed the conformation of the V3 loop in clade C viruses. It was shown that intrinsic differences in V3 conformation exist between clade B and C which are related to the sequence variability in the stem and turn regions, but not in the base of the V3 loop (Patel et al., 2008). Different V3 conformations may have consequences on the antibody response, with less cross-reactivity and limited evolution of the V3 region in the clade C viruses towards evolving X4-tropic variant loops (Patel et al., 2008). Indeed, clade C envelopes carry a relatively conserved V3 sequence, and X4 variants of clade C evolve less frequently compared with clade B (Morris et al., 2001; Peeters et al., 1999).

Another study visualized the dynamic 3D structure of the V3 crown by ab initio folding and suggested a rigid, not beta-hairpin-like, structure of the V3 loop crown in the consensus cladeC V3 sequence compared to the flexible beta-hairpin structure in consensus clade B V3 (Almond et al., 2012). This conformational alteration may impart immune evasion to the virus but, conversely, antibodies specific to consensus clade C V3 would be less effective against other, more beta-hairpin-like strains.

In conclusion, it was found that human anti-V3 mAbs derived from Cameroonian patients infected mainly with clade AG displayed higher relative binding affinity to various recombinant gp120s and significantly higher neutralizing activities than V3 mAbs from Indian patients infected mostly by clade C HIV-1. Particularly interesting is the fact that among anti-V3 mAbs encoded by the VH5-51/VL lambda genes, those mAbs from Cameroon have a higher percentage of mutations and displayed significantly more efficient neutralization than those from India. These results suggest the clade C V3 region may have lower immunogenicity compared to clade AG V3 loop from Cameroon and its sequence can serve as a plausible template for vaccine immunogen design.

Materials and Methods

Monoclonal antibodies

Eighteen human anti-V3 mAbs developed from non-B clade infected individuals living in Cameroon and India were used in this study (Gorny et al., 2004;2009;2006). All donors were chronically HIV-1 infected of unknown duration. Five of these non-B clade V3 mAbs, 4139, 4210, 4487, 4490 and 4508 (Table 1), were recently developed using cellular techniques as previously described with some modifications (Gorny et al., 1991). Briefly, peripheral blood mononuclear cells (PBMC) were transformed with Epstein-Barr virus (EBV) and cultured with a polyclonal B cell activator, CpG (Traggiai et al., 2004) which enhances EBV infection and B cell transformation. The culture supernatants were screened for binding activity to V3-cholera toxin B (CTB) fusion protein (Totrov et al., 2010) containing consensus V3 sequences from clade B and C. The reactive cells were fused with the heteromyeloma cell line SHM-D33 (Teng et al., 1983) and the resulting hybridoma cells that continued to make functional V3 Abs were repeatedly cloned until monoclonality was achieved.

In addition, one anti-V3 mAb 447-52D, generated from a clade B infected individual (Gorny et al., 1993), and one control mAb, 1418, specific to parvovirus B19 (Gigler et al., 1999) were also tested as controls (Table 1).

Envelope proteins

Seventeen recombinant gp120s representing sequences of primary HIV-1 isolates from clade A, B and C (produced in 293 cells) were purchased from Immune Technology Corp. (http://www.immune-tech.com); gp120MN (clade B) was purchased from Immunodiagnostics, Inc. (Woburn, MA).

RT-PCR amplification of the Ig variable region of the heavy and light chain genes

The nucleotide sequence of the Ig variable genes and gene usage by the newly developed human anti-V3 mAbs was determined as previously described (Gorny et al., 2011;2009). Briefly, messenger RNA was extracted from the hybridoma cell lines producing anti-V3 mAbs and reverse transcribed into cDNA using oligo dT primer. The variable domain of the heavy and lambda light chain genes (VH and VL) was amplified from poly-C tailed cDNA by PCR using deoxyinosine-containing anchor primer as the forward primer (Invitrogen) and gene specific primer as the reverse primer located in the constant region of VH family genes, κ and/or λ genes. PCR amplification was performed using a cycling program and ethidium bromidestained 0.8% agarose gels were used to visualize the PCR products. The bands of appropriate size were excised, purified and then cloned into the 2.1-TOPO TA cloning vector (Invitrogen). For each chain, 6 to 12 independent clones were screened. The plasmids with the appropriate inserts were sequenced in both directions using the M13 primers. All sequencing reactions were performed at the Macrogen, Rockville, MD. The sequence data were analyzed using Pregap4, BioEdit softwares and the International ImMunoGene Tics (IMGT) information system (http://imgt.cines.fr).

Binding assay

The binding activity of the anti-V3 mAbs against the gp120s was tested by ELISA as described (Gorny et al., 1997). Briefly, ELISA plates were coated overnight with gp120s at 1.0 μg/ml, blocked with 2% bovine serum albumin in PBS, and then incubated with mAbs at a concentration ranging from 10 to 0.00001 μg/ml. The bound mAbs were detected by incubation with alkaline phosphatase-conjugated goat anti-human IgG (γ specific) (SouthernBiotech, Birmingham, AL) followed by adding substrate to develop color and the plates were read at 410 nm. The relative affinities of mAb binding to gp120s were determined by measuring the concentration of mAbs required for 50% maximal binding defined when the binding curve reached the saturation level as described (Gorny et al., 2000).

TZM-bl neutralization assay

The anti-V3 mAbs were tested for neutralizing activities against 41 psVs using the TZM-bl cell line as target cells, as described (Li et al., 2005; Seaman et al., 2010). Pseudoviruses expressing single cloned envelopes (Env) derived from tier 1 and tier 2 viruses were classified as neutralization-sensitive and -resistant, respectively. Briefly, 2-fold serial dilutions of mAbs, starting from 50 μg/ml, were pre-incubated with the psVs and the mAb/virus mixtures were incubated 48 h with TZM-bl cells expressing CD4, CXCR4 and CCR5. The virus infectivity was determined by measuring the luciferase activity in the cell lysates. The 50% inhibitory concentration of mAb (IC50) was determined at which relative luminescence units were reduced 50% compared to virus control wells.

Statistical analysis

A Chi-Squared test was used for analysis of the binding data and the neutralizing IC50 values of two panels of anti-V3 mAbs, including the separate analysis of tier 1, tier 2, clade B, non-clade B psVs, as well as VH5-51 and non-VH5-51 encoded V3 mAbs. Because some of the mAb/psV or mAb/gp120 combinations failed to react, the neutralizing IC50 and relative affinity data was treated as censored data and therefore the Chi-Squared longrank test was used. The Student’s t-test was used for analysis of mutation rates and relative affinity of V3 mAbs binding to individual gp120s.

Supplementary Material

1

Acknowledgments

This study was supported in part by NIH grants AI091543, AI077451 (MKG), AI083142, and HL59725, by research funds from the Bill and Melinda Gates Foundation and the Department of Veterans Affairs (SZP). Monoclonal antibodies were supplied with the support of the NIH AIDS Research and Reference Reagent Program.

Footnotes

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2012.12.010.

References

  1. Almond D, Krachmarov C, Swetnam J, Zolla-Pazner S, Cardozo T. Resistance of subtype C HIV-1 strains to anti-V3 loop antibodies. Adv Virol. 2012;2012:803535. doi: 10.1155/2012/803535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andrus L, Prince AM, Bernal I, McCormack P, Lee DH, Gorny MK, Zolla-Pazner S. Passive immunization with a human immunodeficiency virus type-1 neutralizing monoclonal antibody in Hu-PBL-SCID mice: isolation of a neutralization escape variant. J Infect Dis. 1998;177:889–897. doi: 10.1086/515251. [DOI] [PubMed] [Google Scholar]
  3. Corti D, Langedijk JP, Hinz A, Seaman MS, Vanzetta F, Fernandez-Rodriguez BM, Silacci C, Pinna D, Jarrossay D, Balla-Jhagjhoorsingh S, Willems B, Zekveld MJ, Dreja H, O’Sullivan E, Pade C, Orkin C, Jeffs SA, Montefiori DC, Davis D, Weissenhorn W, McKnight A, Heeney JL, Sallusto F, Sattentau QJ, Weiss RA, Lanzavecchia A. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. PloS One. 2010;5:e8805. doi: 10.1371/journal.pone.0008805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Emini EA, Schleif WA, Nunberg JH, Conley AJ, Eda Y, Tokiyoshi S, Putney SD, Matsushita S, Cobb KE, Jett CM, Eichberg JW, Murthy KK. Prevention of HIV-1 infection in chimpanzees by gp120 V3 domain-specific monoclonal antibody. Nature. 1992;355:728–730. doi: 10.1038/355728a0. [DOI] [PubMed] [Google Scholar]
  5. Gigler A, Dorsch S, Hemauer A, Williams C, Kim S, Young NS, Zolla-Pazner S, Wolf H, Gorny MK, Modrow S. Generation of neutralizing human monoclonal antibodies against parvovirus B19 proteins. J Virol. 1999;73:1974–1979. doi: 10.1128/jvi.73.3.1974-1979.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gorny MK, Revesz K, Williams C, Volsky B, Louder MK, Anyangwe CA, Krachmarov CP, Kayman SC, Pinter A, Nadas A, Nyambi PN, Mascola JR, Zolla-Pazner S. The V3 loop is accessible on the surface of most human immunodeficiency virus type 1 primary isolates and serves as a neutralization epitope. J Virol. 2004;78:2394–2404. doi: 10.1128/JVI.78.5.2394-2404.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gorny MK, Sampson J, Li H, Jiang X, Totrov M, Wang X-H, Williams C, O’Neal T, Volsky B, Li L, Cardozo T, Nyambi P, Zolla-Pazner S, Kong X-P. Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5-51/VL lambda genes define a conserved antigenic structure. PloS One. 2011;6:e27780. doi: 10.1371/journal.pone.0027780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gorny MK, VanCott TC, Hioe C, Israel ZR, Michael NL, Conley AJ, Williams C, Kessler JA, 2nd, Chigurupati P, Burda S, Zolla-Pazner S. Human monoclonal antibodies to the V3 loop of HIV-1 with intra- and interclade cross-reactivity. J Immunol. 1997;159:5114–5122. [PubMed] [Google Scholar]
  9. Gorny MK, VanCott TC, Williams C, Revesz K, Zolla-Pazner S. Effects of oligomerization on the epitopes of the human immunodeficiency virus type 1 envelope glycoproteins. Virology. 2000;267:220–228. doi: 10.1006/viro.1999.0095. [DOI] [PubMed] [Google Scholar]
  10. Gorny MK, Wang XH, Williams C, Volsky B, Revesz K, Witover B, Burda S, Urbanski M, Nyambi P, Krachmarov C, Pinter A, Zolla-Pazner S, Nadas A. Preferential use of the VH5-51 gene segment by the human immune response to code for antibodies against the V3 domain of HIV-1. Mol Immunol. 2009;46:917–926. doi: 10.1016/j.molimm.2008.09.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gorny MK, Williams C, Volsky B, Revesz K, Wang XH, Burda S, Kimura T, Koning FA, Nadas A, Anyangwe C, Nyambi P, Krachmarov C, Pinter A, Zolla-Pazner S. Cross-clade neutralizing activity of human anti-V3 monoclonal antibodies derived from the cells of individuals infected with non-B clades of HIV-1. J Virol. 2006;80:6865–6872. doi: 10.1128/JVI.02202-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gorny MK, Xu J-Y, Gianakakos V, Karwowska S, Williams C, Sheppard HW, Hanson CV, Zolla-Pazner S. Production of site-selected neutralizing human monoclonal antibodies against the third variable domain of the HIV-1 envelope glycoprotein. Proc Natl Acad Sci USA. 1991;88:3238–3242. doi: 10.1073/pnas.88.8.3238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gorny MK, Xu J-Y, Karwowska S, Buchbinder A, Zolla-Pazner S. Repertoire of neutralizing human monoclonal antibodies specific for the V3 domain of HIV-1 gp120. J Immunol. 1993;150:635–643. [PubMed] [Google Scholar]
  14. Haynes BF, Gilbert PB, McElrath MJ, Zolla-Pazner S, Tomaras GD, Alam SM, Evans DT, Montefiori DC, Karnasuta C, Sutthent R, Liao HX, DeVico AL, Lewis GK, Williams C, Pinter A, Fong Y, Janes H, DeCamp A, Huang Y, Rao M, Billings E, Karasavvas N, Robb ML, Ngauy V, de Souza MS, Paris R, Ferrari G, Bailer RT, Soderberg KA, Andrews C, Berman PW, Frahm N, De Rosa SC, Alpert MD, Yates NL, Shen X, Koup RA, Pitisuttithum P, Kaewkungwal J, Nitayaphan S, Rerks-Ngarm S, Michael NL, Kim JH. Immune-correlates analysis of an HIV-1 vaccine efficacy trial. N Engl J Med. 2012;366:1275–1286. doi: 10.1056/NEJMoa1113425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hioe CE, Wrin T, Seaman MS, Yu X, Wood B, Self S, Williams C, Gorny MK, Zolla-Pazner S. Anti-V3 monoclonal antibodies display broad neutralizing activities against multiple HIV-1 subtypes. PloS One. 2010;5:e10254. doi: 10.1371/journal.pone.0010254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jiang X, Burke V, Totrov M, Williams C, Cardozo T, Gorny MK, Zolla-Pazner S, Kong X-P. Conserved structural elements in the V3 crown of HIV-1 GP120. Nat Struct Mol Biol. 2010;17:955–961. doi: 10.1038/nsmb.1861. [DOI] [PubMed] [Google Scholar]
  17. Letvin NL, Robinson S, Rohne D, Axthelm MK, Fanton JW, Bilska M, Palker TJ, Liao HX, Haynes BF, Montefiori DC. Vaccine-elicited V3 loop-specific antibodies in rhesus monkeys and control of a simian-human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate envelope. J Virol. 2001;75:4165–4175. doi: 10.1128/JVI.75.9.4165-4175.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li M, Gao F, Mascola JR, Stamatatos L, Polonis VR, Koutsoukos M, Voss G, Goepfert P, Gilbert P, Greene KM, Bilska M, Kothe DL, Salazar-Gonzalez JF, Wei X, Decker JM, Hahn BH, Montefiori DC. Human immunodeficiency virus type 1 env clones from acute and early subtype B Infections for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol. 2005;79:10108–10125. doi: 10.1128/JVI.79.16.10108-10125.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Montefiori DC, Karnasuta C, Huang Y, Ahmed H, Gilbert P, de Souza MS, McLinden R, Tovanabutra S, Laurence-Chenine A, Sanders-Buell E, Moody MA, Bonsignori M, Ochsenbauer C, Kappes J, Tang H, Greene K, Gao H, Labranche CC, Andrews C, Polonis VR, Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Self SG, Berman PW, Francis D, Sinangil F, Lee C, Tartaglia J, Robb ML, Haynes BF, Michael NL, Kim JH. Magnitude and breadth of the neutralizing antibody response in the RV144 and Vax003 HIV-1 vaccine efficacy trials. J Infect Dis. 2012;206:431–441. doi: 10.1093/infdis/jis367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Moody MA, Yates NL, Amos JD, Drinker MS, Eudailey JA, Gurley TC, Marshall G, Whitesides JF, Chen X, Foulger A, Yu JS, Zhang R, Meyerhoff RR, Parks R, Scull JC, Wang L, Vandergrift NA, Pickeral J, Pollara J, Kelsoe G, Alam SM, Ferrari G, Montefiori DC, Voss G, Liao HX, Tomaras GD, Haynes BF. HIV-1 gp120 vaccine induces affinity maturation in both new and persistent antibody clonal lineages. J Virol. 2012;86:7496–7507. doi: 10.1128/JVI.00426-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Morris L, Cilliers T, Bredell H, Phoswa M, Martin DJ. CCR5 is the major coreceptor used by HIV-1 subtype C isolates from patients with active tuberculosis. AIDS Res Hum Retroviruses. 2001;17:697–701. doi: 10.1089/088922201750236979. [DOI] [PubMed] [Google Scholar]
  22. Osmanov S, Pattou C, Walker N, Schwardlander B, Esparza J. Estimated global distribution and regional spread of HIV-1 genetic subtypes in the year 2000. J Acquir Immune Defic Syndr. 2002;29:184–190. doi: 10.1097/00042560-200202010-00013. [DOI] [PubMed] [Google Scholar]
  23. Pantophlet R, Aguilar-Sino RO, Wrin T, Cavacini LA, Burton DR. Analysis of the neutralization breadth of the anti-V3 antibody F425-B4e8 and re-assessment of its epitope fine specificity by scanning mutagenesis. Virology. 2007;364:441–453. doi: 10.1016/j.virol.2007.03.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Patel MB, Hoffman NG, Swanstrom R. Subtype-specific conformational differences within the V3 region of subtype B and subtype C human immunodeficiency virus type 1 Env proteins. J Virol. 2008;82:903–916. doi: 10.1128/JVI.01444-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Peeters M, Vincent R, Perret JL, Lasky M, Patrel D, Liegeois F, Courgnaud V, Seng R, Matton T, Molinier S, Delaporte E. Evidence for differences in MT2 cell tropism according to genetic subtypes of HIV-1: syncytium-inducing variants seem rare among subtype C HIV-1 viruses. J Acquir Immune Defic Syndr Hum Retrovirol. 1999;20:115–121. doi: 10.1097/00042560-199902010-00002. [DOI] [PubMed] [Google Scholar]
  26. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C, de Souza M, Adams E, Benenson M, Gurunathan S, Tartaglia J, McNeil JG, Francis DP, Stablein D, Birx DL, Chunsuttiwat S, Khamboonruang C, Thongcharoen P, Robb ML, Michael NL, Kunasol P, Kim JH. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med. 2009;361:2209–2220. doi: 10.1056/NEJMoa0908492. [DOI] [PubMed] [Google Scholar]
  27. Scheid JF, Mouquet H, Feldhahn N, Seaman MS, Velinzon K, Pietzsch J, Ott RG, Anthony RM, Zebroski H, Hurley A, Phogat A, Chakrabarti B, Li Y, Connors M, Pereyra F, Walker BD, Wardemann H, Ho D, Wyatt RT, Mascola JR, Ravetch JV, Nussenzweig MC. Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals. Nature. 2009;458:636–640. doi: 10.1038/nature07930. [DOI] [PubMed] [Google Scholar]
  28. Scheid JF, Mouquet H, Ueberheide B, Diskin R, Klein F, Olivera TY, Pietzsch J, Fenyo D, Abadir A, Velinzon K, Hurley A, Myung S, Boulad F, Poignard P, Burton D, Pereyra F, Ho DD, Walker BD, Seaman MS, Bjorkman PJ, Chait BT, Nussenzweig MC. Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science. 2011;333:1633–1637. doi: 10.1126/science.1207227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Seaman MS, Janes H, Hawkins N, Grandpre LE, Devoy C, Giri A, Coffey RT, Harris L, Wood B, Daniels MG, Bhattacharya T, Lapedes A, Polonis VR, McCutchan FE, Gilbert PB, Self SG, Korber BT, Montefiori DC, Mascola JR. Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. J Virol. 2010;84:1439–1452. doi: 10.1128/JVI.02108-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Teng NN, Lam KS, Calvo Riera F, Kaplan HS. Construction and testing of mouse-human heteromyelomas for human monoclonal antibody production. Proc Natl Acad Sci USA. 1983;80:7308–7312. doi: 10.1073/pnas.80.23.7308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tiller T, Tsuiji M, Yurasov S, Velinzon K, Nussenzweig MC, Wardemann H. Autoreactivity in human IgG+ memory B cells. Immunity. 2007;26:205–213. doi: 10.1016/j.immuni.2007.01.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Totrov M, Jiang X, Kong XP, Cohen S, Krachmarov C, Salomon A, Williams C, Seaman MS, Cardozo T, Gorny MK, Wang S, Lu S, Pinter A, Zolla-Pazner S. Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold. Virology. 2010;405:513–523. doi: 10.1016/j.virol.2010.06.027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Traggiai E, Becker S, Subbarao K, Kolesnikova L, Uematsu Y, Gismondo MR, Murphy BR, Rappuoli R, Lanzavecchia A. An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus. Nat Med. 2004;10:871–875. doi: 10.1038/nm1080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Walker LM, Huber M, Doores KJ, Falkowska E, Pejchal R, Julien JP, Wang SK, Ramos A, Chan-Hui PY, Moyle M, Mitcham JL, Hammond PW, Olsen OA, Phung P, Fling S, Wong CH, Phogat S, Wrin T, Simek MD, Koff WC, Wilson IA, Burton DR, Poignard P Principal Investigators, P.G. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature. 2011;477:466–470. doi: 10.1038/nature10373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, Goss JL, Wrin T, Simek MD, Fling S, Mitcham JL, Lehrman JK, Priddy FH, Olsen OA, Frey SM, Hammond PW, Miiro G, Serwanga J, Pozniak A, McPhee D, Manigart O, Mwananyanda L, Karita E, Inwoley A, Jaoko W, Dehovitz J, Bekker LG, Pitisuttithum P, Paris R, Allen S, Kaminsky S, Zamb T, Moyle M, Koff WC, Poignard P, Burton DR. Broad and potent neutralizing antibodies from an african donor reveal a new HIV-1 vaccine target. Science. 2009;326:285–289. doi: 10.1126/science.1178746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Watkins JD, Siddappa NB, Lakhashe SK, Humbert M, Sholukh A, Hemashettar G, Wong YL, Yoon JK, Wang W, Novembre FJ, Villinger F, Ibegbu C, Patel K, Corti D, Agatic G, Vanzetta F, Bianchi S, Heeney JL, Sallusto F, Lanzavecchia A, Ruprecht RM. An anti-HIV-1 V3 loop antibody fully protects cross-clade and elicits T-cell immunity in macaques mucosally challenged with an R5 clade C SHIV. PloS One. 2011;6:e18207. doi: 10.1371/journal.pone.0018207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wu X, Yang ZY, Li Y, Hogerkorp CM, Schief WR, Seaman MS, Zhou T, Schmidt SD, Wu L, Xu L, Longo NS, McKee K, O’Dell S, Louder MK, Wycuff DL, Feng Y, Nason M, Doria-Rose N, Connors M, Kwong PD, Roederer M, Wyatt RT, Nabel GJ, Mascola JR. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science. 2010;329:856–861. doi: 10.1126/science.1187659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zolla-Pazner S, Kong X, Jiang X, Cardozo T, Nadas A, Cohen S, Totrov M, Seaman MS, Wang S, Lu S. Cross-clade HIV-1 neutralizing antibodies induced with V3-scaffold protein immunogens following priming with gp120 DNA. J Virol. 2011;85:9887–9898. doi: 10.1128/JVI.05086-11. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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NIHMS451797-supplement-1.doc (890KB, doc)

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