HIV-2 neutralization by intact V3-specific Fab fragments
© Sourial and Nilsson; licensee BioMed Central Ltd. 2008
Received: 03 March 2008
Accepted: 18 August 2008
Published: 18 August 2008
The V3 region of both HIV-1 gp120 and HIV-2 gp125 surface glycoprotein has been described as a target for neutralizing antibodies. In this study a conformation-sensitive (3C4) and a linear site-specific (7C8) anti-HIV-2 V3 monoclonal antibody (mAb) were characterized. The neutralization capacity of the purified mAbs and their respective papain-generated Fab fragments was analyzed. The Fabs were further characterized by sequence analysis. Our results demonstrate that neither purified mAbs were capable of neutralizing HIV-2, while intact Fab fragments from both mAbs blocked in vitro infection of HIV-2 isolates. Moreover, the conformation sensitive 3C4 Fab neutralized both subtype A and B HIV-2 isolates and SIVsm. Sequence analysis of the hypervariable regions of 3C4 Fab and 7C8 Fab revealed that the third CDR of the heavy chain (CDRH3) of the antibodies was not as long as many of the previously characterized neutralizing antibodies. Our findings suggest that whole 7C8 and 3C4 mAbs are sterically hindered from neutralizing HIV-2, whereas the smaller size of Fab fragments enables access to the V3 region on the virion surface.
The HIV-1 V3 region has been identified as a target site recognized by neutralizing antibodies . Monoclonal antibodies (mAbs) targeting conformational epitopes within the V3 region have been demonstrated to neutralize primary HIV-1 isolates [2–5]. Furthermore, anti-HIV-1 V3-specific mAbs have recently been shown to have broad cross-reactivity, which was dependent on the extent of masking of the V1/V2 regions and the sequence at the crown of the V3-loop .
Neutralizing antibodies have been reported to bind within the HIV-2 V3 region [7, 8], with the FHSQ residues at the tip of the V3-loop . However, mAbs recognizing the linear (FHSQ) site on gp125 could not neutralize HIV-2 isolates [9, 10]. Conversely, a conformational epitope composed of FHSQ (amino acids 315–318 in HIV-2 ISY clone) and WCR (amino acids 329–331) has been described in the V3 region of gp125 [9, 11], where mAbs recognizing conformational epitopes in the gp125 V3 region could neutralize HIV-2 isolates [9, 12]. While the exposure of the V3 region in HIV-1 is debated, the accessibility of neutralizing sites on HIV-2 V3 region has not been as extensively characterized as for HIV-1.
Previously, two hybridoma cell-lines have been isolated from mice immunized with two overlapping peptides of HIV-2 spanning the center and C-terminus of the V3 region . The hybridoma expressing the 3C4 mAb recognized both peptides, while the 7C8 mAb recognized only the center of the V3 region. Mouse ascitic fluid containing 3C4 has been reported to neutralize different isolates of HIV-2 at a dilution of 1:20, whereas the mouse ascitic fluid containing 7C8 had no neutralizing effect. Further characterization of the binding of these two mAbs to recombinant gp125 indicated that 3C4 is conformation sensitive while 7C8 binds to a linear site .
In this study, the HIV-2 neutralization capacity of protein A-purified 3C4 and 7C8 mAbs was analyzed. A neutralization assay employing phytohemagglutinin-stimulated PBMCs (peripheral blood mononuclear cells) was used . Two-fold serial dilutions of mAb starting at 100 μg/ml were incubated for one hour at 37°C with a minimum of 15 TCID50 (50% tissue culture infectious dose) tissue culture supernatant from virus infected cells. PBMCs (105) were then added to the mix and incubated overnight at 37°C. The medium was changed with fresh IL-2 containing medium on the following day and on day 4. Seven days after infection, supernatants were collected and analyzed for HIV-2 antigen by a capture ELISA . The neutralization concentration was defined as the concentration where a 80% reduction or more of optical density at 490 nm in the culture supernatant was seen as compare to the negative control (i.e. IC80). To determine the virus inoculum dose, a TCID50 titration was performed in parallel to each neutralization experiment.
Contrary to what has been reported previously for 3C4, up to 100 μg/ml of the purified mAbs did not neutralize any of the HIV-2 isolates tested (data not shown). This could be explained by the fact that ascitic fluid contains > 10 mg/ml of mAb as well as other components that could alter the neutralization effect.
In vitro virus neutralization exhibited by 7C8 Fab and 3C4 Fab fragments given as the concentration of Fab needed for IC80 neutralization.
CCR1, CCR3, CCR5
The mechanism underlying the more potent neutralizing effect of Fab fragments has been previously correlated with the smaller size of Fab fragments compared to the size of whole IgG, where steric factors could limit the accessibility to neutralizing epitopes . Previous reports have also described different neutralization mechanisms between mAbs and Fabs at the attachment or fusion of the virus with target cells [20, 21].
To characterize the Fab fragments, the variable regions of 7C8 and 3C4 were sequenced according to their subtypes. Sub-typing of 7C8 and 3C4 revealed that both mAbs have kappa light chains and that they belong to subtypes IgG1 and IgG2a, respectively. Alignment of 7C8 and 3C4 variable regions (Figure 1B) indicated that the light chains of both Fabs are more conserved (93% identity) than the heavy chain (53% identity). The predicted CDR loops are indicated in Figure 1B, where the CDR3 loop of 7C8 and 3C4 heavy chains consists of 13 and 11 residues, respectively.
The third CDR of the heavy chain (CDRH3) of an antibody plays a distinctive role in determining antibody specificity . Sequence analysis of 7C8 CDRH3 revealed a comparatively long region of 13 amino acids (aa), whereas the average length of CDRH3 in mice is between 8 and 9 aa and <11% of mice antibodies have a CDRH3 length of >13 aa [22, 23]. Most human anti-gp120 antibodies have CDRH3 of >15 aa , as exemplified by the neutralizing mAbs/Fab, 17b, b12, 2F5, and X5 which recognize different epitopes on gp120 and have CDRH3 lengths of 19, 18, 22, and 22 aa respectively [25–29]. Therefore, we do not believe that the comparatively longer CDRH3 regions of 3C4 and 7C8 would play a strong role in the viral neutralization.
In conclusion, a confirmation sensitive HIV-2 V3 specific Fab was shown to neutralize both HIV-2 A and B subtypes and SIVsm. Further analyses indicate that the smaller size of intact Fab fragments may be a determining factor for HIV-2 V3-specfic viral neutralization. These results could provide clues to small molecule anti-HIV inhibitors.
The study was supported by the Karolinska Institute, Stockholm, Sweden.
- Huber M, Trkola A: Humoral immunity to HIV-1: neutralization and beyond. J Internal Med 2007, 262: 5-25. 10.1111/j.1365-2796.2007.01819.xView ArticlePubMedGoogle Scholar
- Binley JM, Wrin T, Korber B, Zwick MB, Wang M, Chappey C, Steigler G, Kunert R, Zolla-Pazner S, Katinger H, Petropoulos CJ, Burton DR: Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies. J Virol 2004, 78: 13232-13252. 10.1128/JVI.78.23.13232-13252.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Conley AJ, Gorny MK, Kessler JA II, Boots LJ, Ossorio-Castro M, Koenig S, Lineberger DW, Emini EA, Williams C, Zolla-Pazner S: Neutralization of primary human immunodeficiency virus type 1 isolates by the broadly reactive anti-V3 monoclonal antibody, 447-52D. J Virol 1994, 68: 6994-7000.PubMed CentralPubMedGoogle Scholar
- Zolla-Pazner S, Zhong P, Revesz K, Volsky B, Williams C, Nyambi P, Gorny MK: The cross-clade neutralizing activity of a human monoclonal antibody is determined by the GPGR V3 motif of HIV type 1. AIDS Res Hum Retroviruses 2004, 20: 1254-1258. 10.1089/aid.2004.20.1254View ArticlePubMedGoogle Scholar
- Gorny MK, Revesz K, Williams C, Volsky B, Lauder MK, Anyanywe CA, Krachmarov C, 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. 10.1128/JVI.78.5.2394-2404.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Krachmarov CP, Honnen WJ, Kayman SC, Gorney MK, Zolla-Pazner S, Pinter A: Factors determining the breadth and potency of neutralization by V3-specific human monoclonal antibodies derived from subjects infected with clade A and clade B strains of human immunodeficiency virus type 1. J Virol 2006, 80: 7127-7135. 10.1128/JVI.02619-05PubMed CentralView ArticlePubMedGoogle Scholar
- Björling E, Broliden K, Bernardi D, Utter G, Thorstensson R, Chiodi F, Norrby E: Hyperimmune antisera against synthetic peptides representing the glycoprotein of human ummundeficiency virus type 2 can mediate neutralization and antibody-mediated cytotoxic activity. Proc Natl Acad Sci USA 1991, 88: 6082-6086. 10.1073/pnas.88.14.6082PubMed CentralView ArticlePubMedGoogle Scholar
- Matsushita S, Matsumi S, Yoshimura K, Morikita T, Murakami T, Takatsuki K: Neutralizing monoclonal antibodies against human immunodeficiency virus type 2 gp120. J Virol 1995, 69: 3333-3340.PubMed CentralPubMedGoogle Scholar
- Björling E, Chiodi F, Utter G, Norrby E: Two neutralizing domains in the V3 region in the envelope glycoprotein gp125 of HIV type 2. J Immunol 1994, 152: 1952-1959.PubMedGoogle Scholar
- McKnight A, Shotton C, Cordell J, Jones I, Simmons G, Clapham PR: Location, exposure, and conservation of neutralizing and nonneutralizing epitopes on human immunodeficiency virus type 2 SU glycoprotein. J Virol 1996, 70: 4598-4606.PubMed CentralPubMedGoogle Scholar
- Mörner A, Björndal A, Albert J, Kewalramani VN, Littman DR, Inoue R, Thorstensson R, Fenyö EM, Björling E: Primary human immunodeficiency virus type 2 (HIV-2) isolates, like HIV-1 isolates, frequently use CCR5 but show promiscuity in coreceptor usage. J Virol 1999, 73: 2343-2349.PubMed CentralPubMedGoogle Scholar
- McKnight A, Dittmar MT, Moniz-Periera J, Ariyoshi K, Reeves JD, Hibbitts S, Whitby D, Aarons E, Proudfoot AE, Whittle H, Clapham PR: A broad range of chemokine receptors are used by primary isolates of human immunodeficiency virus type 2 as coreceptors with CD4. J Virol 1998, 72: 4065-4071.PubMed CentralPubMedGoogle Scholar
- Sourial S, Nilsson C, Wärnmark A, Achour A, Harris RA: Deletion of the V1/V2 region does not increase the accessibility of the V3 region of recombinant gp125. Curr HIV Res 2006, 4: 229-237. 10.2174/157016206776055066View ArticlePubMedGoogle Scholar
- Lizeng Q, Skott P, Sourial S, Nilsson C, Andersson S, Ehnlund M, Taveira N, Björling E: Serum immunolglobulin A (IgA)-mediated immunity in human immunodeficiency virus type 2 (HIV-2) infection. Virology 2003, 308: 225-232. 10.1016/S0042-6822(02)00088-0View ArticlePubMedGoogle Scholar
- Thorstensson R, Walther L, Putkonen P, Albert J, Biberfeld G: A capture enzyme immunoassay for detection of HIV-2/SIV antigen. J Acquir Immune Defic Syndr 1991,4(4):374-379.PubMedGoogle Scholar
- Labrijn AF, Poignard P, Raja A, Zwick MB, Delgado K, Franti M, Binley J, Vivona V, Grundner C, Huang CC, Venturi M, Petropoulos CJ, Wrin T, Dimitrov DS, Robinson J, Kwong PD, Wyatt RT, Sodroski J, Burton DR: Access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp120 is sterically restricted on primary human immunodeficiency virus type 1. J Virol 2003, 77: 10557-10565. 10.1128/JVI.77.19.10557-10565.2003PubMed CentralView ArticlePubMedGoogle Scholar
- Roben P, Moore JP, Thali M, Sodroski J, Barbas CF 3rd, Burton DR: Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. J Virol 1994, 68: 4821-4828.PubMed CentralPubMedGoogle Scholar
- Parham P: Preparation and purification of active fragments from mouse monoclonal antibodies. In Cellular Immunology. Volume 1. 4th edition. Edited by: Weir DM. California: Blackwell Scientific Publications; 1986.
- Ornatowska M, Glasel JA: Direct production of Fv-fragments from a family of monoclonal IgGs papain digestion. Mol Immunol 1991, 28: 383-391. 10.1016/0161-5890(91)90151-9View ArticlePubMedGoogle Scholar
- McInerney TL, McLain L, Armstrong SJ, Dimmock NJ: A human IgG1 (b12) specific for the CD4 binding site of HIV-1 neutralizes by inhibiting the virus fusion entry process, but b12 Fab neutralizes by inhibiting a postfusion event. Virology 1997, 233: 313-26. 10.1006/viro.1997.8547View ArticlePubMedGoogle Scholar
- Edwards MJ, Dimmock NJ: Two influenza A virus-specific Fabs neutralize by inhibiting virus attachment to target cells, while neutralization by their IgGs is complex and occurs simultaneously through fusion inhibition and attachment inhibition. Virology 2000, 278: 423-435. 10.1006/viro.2000.0631View ArticlePubMedGoogle Scholar
- Popkov M, Mage RG, Alexander CB, Thundivalappil S, Barbas CF: Rabbit immune repertoires as sources for therapeutic monoclonal antibodies: the impact of kappa allotype-correlated variation in cysteine content on antibody libraries selected by phage display. J Mol Biol 2003, 325: 325-335. 10.1016/S0022-2836(02)01232-9View ArticlePubMedGoogle Scholar
- Wu TT, Johnson G, Kabat EA: Length distribution of CDRH3 in antibodies. Proteins 1993, 16: 1-7. 10.1002/prot.340160102View ArticlePubMedGoogle Scholar
- Johnson G, Wu TT: Preferred CDRH3 lengths for antibodies with defined specificities. Int Immunology 1998, 10: 1801-1805. 10.1093/intimm/10.12.1801View ArticleGoogle Scholar
- Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, Hendrickson WA: Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 1998, 393: 648-659. 10.1038/31405View ArticlePubMedGoogle Scholar
- Kwong PD, Wyatt R, Majeed S, Robinson J, Sweet RW, Sodroski J, Hendrickson WA: Structures of HIV-1 gp120 envelope glycoprotein from lab-adapted and primary isolates. Structure 2000, 8: 1329-1339. 10.1016/S0969-2126(00)00547-5View ArticlePubMedGoogle Scholar
- Saphire EO, Parren PW, Pantophlet R, Zwick MB, Morris GM, Rudd PM, Dwek RA, Stanfield RL, Burton DR, Wilson IA: Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design. Science 2001, 293: 1155-1159. 10.1126/science.1061692View ArticlePubMedGoogle Scholar
- Zwick MB, Komori HK, Stanfield RL, Church S, Wang M, Parren PW, Kunert R, Katinger H, Wilson IA, Burton DR: The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5. J Virol 2004, 78: 3155-3161. 10.1128/JVI.78.6.3155-3161.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Dharba R, Phogat S, Labrijn AF, Shu Y, Gu Y, Andrykovitch M, Zhang MY, Pantophlet R, Martin L, Vita C, Burton DR, Dimitrov DS, Ji X: Crystal structure of the broadly cross-reactive HIV-1-neutralizing Fab X5 and fine mapping of its epitope. Biochemistry 2004, 43: 1410-1417. 10.1021/bi035323xView ArticleGoogle Scholar
- Vödrös D, Thorstensson R, Biberfeld G, Schols D, De Clercq E, Fenyo EM: Coreceptor usage of sequential isolates from cynomolgus monkeys experimentally infected with simian immunodeficiency virus (SIVsm). Virology 2001, 291: 12-21. 10.1006/viro.2001.1164View ArticlePubMedGoogle Scholar
- The international ImMunoGeneTics information system database[http://imgt.cines.fr]
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.