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Apr 13, 2016 - multivalently. Additionally, a recombinant gp120 which binds. 2G12 with high affinity was selected as another immunogen. We did not exp...
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Altering the Specificity of the Antibody Response to HIV gp120 with a Glycoconjugate Antigen Chang-Cheng Liu, Canjia Zhai, Xiu-Jing Zheng, and Xin-Shan Ye* State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xue Yuan Rd No. 38, Beijing 100191, China S Supporting Information *

ABSTRACT: Some conserved glycans on the HIV envelope protein are targets of broadly neutralizing antibodies (bnAbs) of HIV. BnAbs provide a precise definition of broadly neutralizing epitopes on the envelope protein of HIV. These epitopes are promising for vaccine design. Many glycan-related antigens with high affinity to bnAbs have been tested as immunogens in vivo. However, it was found that no bnAb-like antibodies were induced. Vaccination with different immunogens containing the same neutralizing epitope may enhance the affinity maturation of antibodies which focus on the shared epitope. This combined immunization strategy showed great potential in peptide epitope-based vaccine design. However, it has not yet been explored on glycan-related epitopes to date. Herein, we take 2G12 as a model to validate this strategy on glycan-related epitopes. A high-affinity antigen of 2G12 was constructed by conjugating the D1 arm tetramannoside to bovine serum albumin. Then, the glycoconjugate was coimmunized with a recombinant gp120, which was expected to selectively benefit the induction of antibodies recognizing the neutralizing epitope of 2G12 on gp120. Mice were inoculated with the two antigens simultaneously or alternately to determine the suitable regimen for this strategy. The serological assays demonstrated that the antibody titers and subtypes responded to the whole gp120 were not improved, and the proportion of antibodies competitively bound to the 2G12 epitope was not enhanced significantly either. However, the coimmunized glycoconjugate selectively raised the proportion of antibodies recognizing D1 arm tetramannoside-related structures on gp120. These results provide important experience for the design of glycan-dependent bnAb-based vaccines.

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unconventional domain-exchange conformation of the heavy chain, providing high-affinity interaction with carbohydrate epitopes.13 Many vaccines have been designed and tested by now. However, they failed to induce 2G12-like antibodies in vivo.21−25 One possible explanation for the failure is the poor antigenicity of these vaccines. The Man9GlcNAc2 or the truncated D1 arm Man4 (1, Figure 1) moieties were multivalently linked to various scaffolds, but the 2G12-affinity was far weaker than that of gp120, as the conformation and orientation of the 2G12 epitopes were not well mimicked. Recently, high 2G12-affinity antigens were obtained by selection from a library of glycoclusters.26 These glycopeptides are worthwhile to be tested in vivo. However, high antigenicity alone is still not sufficient to elicit bnAb-like antibodies in the past efforts on other kinds of antigens, indicating that antigenicity does not necessarily lead to immunogenicity.27 New strategies should be considered to cross the antigenicity− immunogenicity barrier.

he HIV-1 envelope glycoprotein gp120 is heavily glycosylated by N-glycans, typically containing about 25 potential N-glycosylation sites. These glycans contribute about half of the gp120 mass, independently of viral clade.1 The “glycan shield” benefits the virus in various aspects. First, the dense array of the glycans can mask the conserved protein epitopes.2−5 Second, because the glycans are expressed by the host glycosylation machinery, they are considered self-antigens by the immune system and possess low immunogenicity.6,7 Third, the glycans bind to the cell surface receptors such as CD4 and DC-SIGN, facilitating the infection and transmission of the virus.8−10 Although the gp120 glycosylation is tremendously heterogeneous, some glycans are relatively conserved and can be recognized by broadly neutralizing antibodies (bnAbs) such as 2G12,11−14 PG9,15 PG16,16 and PGT128.17 BnAbs, usually separated from “elite neutralizers” with chronic HIV infection, can neutralize a broad range of HIV isolates in vitro and protect the animal models from virus challenge. Therefore, the epitopes of bnAbs are promising targets for HIV-1 vaccine.18,19 The 2G12 antibody, the first isolated glycan-dependent bnAb, mainly binds to several high-mannose glycans (Man9GlcNAc2) on Asn295, Asn332, Asn339, and Asn392 of gp120.11,12,20 The binding surface of antigen is extended by an © XXXX American Chemical Society

Received: March 10, 2016 Accepted: April 13, 2016

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variants benefited the selection of cross-reactive antibodies, which was verified in mice.35 Herein, we choose the most extensively studied 2G12 as a model to explore the combined immunization strategy for glycan-dependent bnAb-based vaccine design. A highly antigenic immunogen of 2G12 was constructed by conjugating the D1 arm tetramannoside to bovine serum albumin (BSA) multivalently. Additionally, a recombinant gp120 which binds 2G12 with high affinity was selected as another immunogen. We did not expect to induce the extraordinary domainexchange 2G12-like antibodies because earlier works showed that the kind of glycoconjugates we used could not bind the germline variant of 2G12.36 Our goal is to induce antibodies recognizing the shared neutralizing epitope of 2G12 on gp120 with the assistance of glycoconjugates. Mice were immunized with the immunogens simultaneously or alternately to investigate the suitable regimen of the combined immunization strategy. We investigated the serum IgG titers of Man4-specific and gp120-reactive antibodies, the subtypes, and the timevarying amount of the gp120-reactive antibodies to study the effects on the immune response of coimmunized immunogens to each other. Using a competitive inhibition test with the 2G12 antibody, the D1 arm tetramannoside, and a triantennary pentamannoside, we showed the directional functions of the coimmunized glycoconjugates on specific glycan moieties of gp120. Our study first proves that the combined immunization strategy is applicable for glycan-dependent bnAb-based vaccine design. On the basis of these results, some key points of designing glycan-dependent bnAb-based vaccines by the combined immunization strategy were proposed.

Figure 1. Structure of D1 arm tetramannoside 1 of the Man9GlcNAc2 moiety and the synthesis of tetramannoside 2 by preactivation-based iterative one-pot strategy.

Affinity maturation of antibodies takes place in germinal centers, where the follicular dendritic cells present antigens to B cells. Somatic hypermutation results in a diverse repertoire of B cells. Some of those B cells can internalize antigens through a B cell receptor (BCR) process and then present the antigens for binding to major histocompatibility complex (MHC) molecules to follicular helper T (Tfh) cells and get survival signals meanwhile.28 The amount of antigens that are internalized by B cells relies on the binding affinity of antigens and BCR. The more antigens the B cells internalize, the more opportunity there is that the B cells are helped by Tfh cells.29 The antigen affinity of BCR is greatly enhanced during this Darwinian evolutionary process. Based on the knowledge of the affinity maturation process, the combined immunization strategy was proposed, which means immunization with different antigen variants containing a same epitope. Those B cells that recognize the shared epitope could internalize all of the antigens and survive with adequate and constant Tfh cells’ help, whereas other B cells could only internalize one of these antigens and do not have a competitive advantage during the affinity maturation period. In this way, the immune response may focus on the shared epitope, and the antigenicity of the antigens may be transformed to immunogenicity in vivo. In fact, the affinity maturation process of bnAbs to HIV has been proved to be extensively directed by the rapidly changed coevolving virus.30,31 The germline predecessors of bnAbs may survive by getting constant stimulation of conserved epitopes on escape mutants; meanwhile the “antivirus spectra” of bnAbs can be also enhanced during this period. This combined immunization strategy was applied to the design of the 2F5-based vaccine.32,33 Several immunogens were designed with the computational method by which the 2F5 epitopes were transplanted to protein scaffolds and then tested in a prime-boosting regimen in vivo. The elicited antibodies had similar antigen combining sites to those of 2F5. Heterotrimeric gp140 immunogens derived from several subtypes of HIV-1 were tested in rhesus macaques in a cocktail regimen and elicited an improved neutralizing antibody response compared with monovalent gp140.34 An in silico model revealed that sequential immunization with immunogen

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RESULTS AND DISCUSSION Design and Preparation of the Highly Antigenic Immunogen of 2G12. 2G12 recognizes a cluster of oligomannosides on the gp120.11,12,20 A crystal structure of the 2G12 fragment of antigen binding (Fab) in complex with Man9GlcNAc2 highlighted the importance of the D1 arm tetramannoside of the Man9GlcNAc2 moiety in 2G12 binding.13 Microarray analysis of several oligomannoside derivatives showed that this tetramannoside exhibited comparable binding affinity with 2G12 to that of Man9.14 Therefore, the D1 arm Man4 was used in many 2G12-based vaccines. Unfortunately, however, no gp120 cross-reactive antibodies were elicited in vivo.21−23,37 On the other hand, when coimmunized with gp120, the high 2G12-affinity of this structure may selectively benefit the affinity maturation of the gp120-reactive antibodies which recognize the epitopes of 2G12. For this reason, we still chose the Man4 structure in our immunogen design. Moreover, the Man4 structure is nonself to the human immune system.38 After being processed by B cells, the carbohydrate epitopes may be presented in conjunction with peptides derived from carrier protein to carbohydrate-specific CD4+ T cells and then get T cells’ help.39 The nonself feature of Man4 may help to bypass the tolerance mechanism of the self Man9GlcNAc2 epitope on gp120. We synthesized the Man4 structure 2 with a linker for protein conjugation by an efficient preactivation-based iterative one-pot strategy40 in good yield (Figure 1). Afterward, the tetramannoside 2 was fully deprotected under the standard conditions to get the target D1 arm tetrasaccharide (Scheme S8, see Supporting Information). BSA was chosen as a carrier protein because previous work has shown that BSA multivalently conjugated by Man4 could induce a strong immune response in rabbits.21 Therefore, 45B

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the 2G12 affinity of BSA-(Man4)19 was far weaker than that of recombinant gp120JR‑CSF. Immunization of the Mice with a Recombinant gp120 and Glycoconjugate BSA-(Man4)19. A group of seven female BALB/c mice of 6−8 weeks old were immunized four times at biweekly intervals with BSA-(Man4)19 and recombinant gp120JR‑CSF simultaneously. It has been revealed that the glycosylation of recombinant monomer gp120 was different from the native envelope trimer, of which the glycans were dominantly high-mannose-type oligosaccharides.41 However, the 2G12 epitope on gp120JR‑CSF generated from the 293 cell line was not obviously affected as the 2G12-affinity of gp120JR‑CSF was still at the nanomolar level (Figure 2C). Therefore, the employment of this gp120 variant was reasonable. In order to exclude the influence of the carrier protein and linker and to demonstrate the T cells’ help for affinity maturation provided by BSA-(Man4)19, an equal molar quantity of BSA-linker22 (Figure 2B) and Man4 1 was used to be coimmunized with gp120JR‑CSF, because the unconjugated Man4 could not be presented and the gp120-elicited B cells, which recognize the epitopes of 2G12, could not internalize the BSA-linker22. Two groups of mice were immunized with gp120JR‑CSF or BSA-(Man4)19 alone. A blank control with PBS buffer was also included. To explore the immunization regimen of the combined immunization strategy, mice were immunized alternately with gp120JR‑CSF and BSA-(Man4)19 for eight times at weekly intervals. The BSA-(Man4)19 was replaced by BSAlinker22 and Man4 1 or by PBS buffer in parallel groups. The mice were kept for 24 weeks and bled eight times. All animals remained healthy during the study, and no adverse reactions were observed. The whole immunization regimen and dosing and bleeding schedule are shown in Figure 3. Detection of the Antibody Titers against Recombinant gp120JR‑CSF and Glycoconjugate OVA-(Man4)10. The fourth bleeding sera had the highest antibody titer, while the eighth bleeding sera could reflect the long-term antibody

or 135-fold molar excesses of Man4 were reacted with the alkyne-modified BSA by copper-catalyzed azide−alkyne cycloaddition (Scheme S15).22,23 On average, 8 or 19 copies of Man4 were conjugated to one BSA molecule, which was confirmed by MALDI-TOF mass spectrometry and SDS-PAGE (Figures S1 and S3). The two glycoconjugates, named BSA-(Man4)8 and BSA-(Man4)19, were tested in parallel with Man4 1 and recombinant gp120 from HIV-1 strain JR-CSF (gp120JR‑CSF) generated by the 293 cell line for their competitive inhibition ability of 2G12 binding to gp120JR‑CSF (Figure 2). It was found

Figure 2. Structures of antigens used in mice immunization and their binding affinity to 2G12. (A) The structure of glycoconjugate BSA(Man4)19. (B) The control antigen BSA-linker22 without Man4 on the BSA conjugate. The number of Man4 or linker was determined by MALDI-TOF mass spectrometry and SDS-PAGE. See Figures S1 and S3 in the Supporting Information. (C) The molar 50% inhibitory concentration (IC50) values of Man4, glycoconjugates and recombinant gp120, determined by competitive ELISA test with gp120 as the coating antigen. The IC50 values are means ± SEM from three separate experiments.

that the molar 50% inhibitory concentration (IC50) value of BSA-(Man4)19 was approximately 7-fold less than that of BSA(Man4)8, probably due to two factors: a higher density display of Man4 and the ability to bind more than one 2G12. However,

Figure 3. Immunization groups and dosing (A) or bleeding (B) schedule for mice experiments. C

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Figure 4. Antibody titers against glycoconjugate OVA-(Man4)10 or recombinant gp120JR‑CSF. IgG antibody titer against OVA-(Man4)10 from the fourth bleeding (A) or the eighth bleeding (B). IgG antibody titer against recombinant gp120JR‑CSF from the fourth bleeding (C) or the eighth bleeding (D). Each dot represents the ELISA result of an individual mouse, and each black line represents the median antibody level of a group of seven mice.

simultaneously (groups 1, 2), were lower than that in the group immunized with gp120JR‑CSF alone (group 3). It might result from the BSA-(Man4)19 or BSA-linker22 having dispersed the immune response to gp120JR‑CSF. However, in the alternate regimen, the coimmunized BSA-(Man4)19 or BSA-linker22 did not induce fewer antibodies. These results suggested that the coimmunogens might bring different influence depending on dosing regimen. No cross-reactive antibodies were detected in group 4, which is consistent with the previous report.21 High antibody titers of gp120JR‑CSF proved that the monomer recombinant gp120JR‑CSF was very immunogenic. However, according to the literature, a large proportion of the antibodies might be induced by unexposed sites of the native trimer or immunodominant sites, thus possessing no broadly neutralizing potency.34,42 The antibody titers against gp120JR‑CSF were higher than that against OVA-(Man4)10, for only Man4-specific antibodies were detected in the ELISA test using OVA(Man4)10 as the coating antigen. To further investigate the influence of the coimmunized glycoconjugate on the immune response of the whole gp120JR‑CSF, we tested the subtypes of the antibodies against gp120JR‑CSF from the last bleeding (Figure 5A). The IgG antibodies were dominantly IgG1 subtype, and the proportion of each subtype in each group was similar. This indicated that the gp120JR‑CSF induced a Th2-polarized immune response in vivo, which was not influenced by the coimmunogens. The antibody titers increased during the immunization, and the highest antibody titers were detected after the last immunization. Afterward, the amount of antibodies declined over time with a similar speed in all groups (Figure 5B). These results indicated that the coimmunogens could not help to

response. Therefore, the immune sera of the fourth and eighth bleeding were used to measure their binding titers against OVA-(Man4)10 (Figure 4, A and B) and gp120JR‑CSF (Figure 4, C and D) by enzyme-linked immunosorbent assay (ELISA). At the beginning, we used BSA-(Man4)19 and BSA-linker22 to detect the Man4-specific antibodies in the presence of 3% (wt/ vol) BSA to absorb the antibodies directed to the protein carrier BSA. The immune sera IgG bound the coating antigen BSA-(Man4)19 very strongly. However, BSA-linker22 also showed moderate binding ability to the serum samples, indicating that some linker-related antibodies were induced in mice. The linker-related antibodies might interfere with the detection of Man4-specific antibodies, even using BSA-linker22 as a control. Therefore, a new coating antigen OVA-(Man4)10 was constructed by conjugating Man4 to an irrelevant protein ovalbumin (OVA) by a different linker, without the triazole ring in BSA-(Man4)19 and BSA-linker22 (Scheme S16). We also created OVA-linker12 (Scheme S16) to use as a blank control in the ELISA test. Both fourth and eighth bleeding immune sera from groups 1, 4, and 6 showed binding ability to OVA(Man4)10, which could be inhibited by Man4 (Figure S6). Immunization with gp120JR‑CSF and BSA-(Man4)19, neither simultaneously nor alternately (group 1 or group 6), induced a higher level of antibodies than immunization with BSA(Man4)19 alone (group 4). High titers of gp120JR‑CSF reactive antibodies were elicited in groups 1, 2, 3, 6, 7, and 8 (Figure 4, C and D). Though antibody titers of these groups did not show statistical difference, several interesting points might be revealed by comparing the median antibody level of each group. The medians of antibody titers of the groups, in which BSA(Man 4 ) 19 or BSA-linker22 and Man 4 were immunized D

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Figure 5. Antibody response against the recombinant gp120JR‑CSF was not improved. (A) The subtypes of IgG antibody against the recombinant gp120JR‑CSF from the eighth bleeding. The immune serum from individual mice was diluted 16 000 times for every subtype. The values are means ± SD of a group of seven mice. (B) The amount of IgG antibody changing over time. The immune serum from individual mice at different times was diluted 16 000 times. Each dot is the mean of OD 490 values of a group of seven mice.

Figure 6. Competitive inhibition test of gp120-reactive antibodies. The immune serum from an individual mouse of the eighth bleeding was diluted 16 000 times. Then, serially diluted 2G12 or IgG1 isotype control (A), Man5 (B), or Man4 (C) were added. The percentage of serum binding of individual mice was calculated as dividing the OD490 of the serum sample with a competitive inhibitor by the OD490 of serum only (B and C) or with isotype control (A). The difference of % serum binding values at the two highest concentrations of competitive inhibitors was not more than 2% for each mouse. Each column showed the mean ± SD of a group of seven mice. Values of groups 1 and 6 marked with asterisks in C were significantly different (P < 0.05) from those of other groups (2, 3, 7, and 8) by the one-way ANOVA, respectively.

induce a more durable immune response to the whole gp120JR‑CSF. Competitive Inhibition Test of gp120-Reactive Antibodies. Considering that a large proportion of gp120-reactive antibodies were directed to undesired epitopes, other tests were required to find out whether the coimmunogens affected the immune response to the 2G12 epitopes. Serially diluted 2G12 or human IgG1 isotype control was added to the diluted serum samples from the last bleeding to competitively bind to the gp120JR‑CSF coated on the plate. 2G12 would inhibit the binding of serum antibodies whose epitopes were overlapped with that of 2G12. The immune sera were diluted 16 000 times at last. In this concentration, the OD490 values of most serum samples were within the linear interval (0 to 2) of our microplate reader. The binding of serum IgG could be weakly inhibited by 2G12 in a dose-dependent manner. The inhibitory effect of 2G12 reached a saturation level when the concentration of 2G12 was around 1 μg mL−1, since almost no more inhibitory effect was detected when the concentration of 2G12 reached 2 μg mL−1. 2G12 elicited slightly higher inhibition for the samples from groups 1 and 6, in which BSA-(Man4)19 was included. This revealed that the coimmunized BSA-(Man4)19 can help to direct the immune response of gp120JR‑CSF to its 2G12 epitope. However, the results were not statistically significant (Figure 6A). We further conducted a competitive inhibition test of gp120reactive antibodies with serially diluted Man4 in a similar protocol. A triantennary pentamannoside (Man5, 39 in Scheme S12) was also involved to confirm whether the inhibition was specific for Man4. The Man5 structure was chosen because it was also present on recombinant gp120 and the Man4-related antibodies could not recognize such a structure.21 The Man5 structure was synthesized via a convergent route in high yield (Scheme S12). The results showed that, even with a very high concentration (4 mg mL−1), the Man5 only caused little inhibition in each group (Figure 6B). The possible explanation could be the relatively rare presence of Man5-type glycans on

the recombinant gp120JR‑CSF and the immunoregulation to selfstructure. The coimmunized BSA-(Man4)19 did not provide any enhancement to the unrelated Man5 epitopes on gp120. Relatively, the Man4 was able to get an average of about 20% reduction of binding in groups 1 and 6, whereas only approximately 10% inhibition was observed in groups 2, 3, 7, and 8 at the maximum concentration of Man4 (Figure 6C). Besides the 2G12 epitope, there are many other high-mannose oligosaccharides terminating with Manα1 → 2Man and/or containing Manα1 → 3Man motifs on recombinant gp120.41 As mentioned above, the 2G12 antibody only caused a slight increase in the percentage of serum binding for groups 1 and 6. Therefore, the coimmunized BSA-(Man4)19 might selectively facilitate the antibody response to Man4-related moieties on gp120JR‑CSF, rather than to the 2G12 epitope only. Conclusions. Our work demonstrated the antibody responses of two immunogens (BSA-(Man4)19 and gp120JR-CSF) sharing a same glycan epitope. The amount of Man4-specific IgG antibodies against OVA-(Man4)10 was similar in all groups in which BSA-(Man4)19 was involved, and the IgG titers of the whole gp120JR‑CSF were not improved by the glycoconjugate. The subtypes of IgG were not changed, and the glycoconjugate could not help to induce a more durable antibody response to the whole gp120JR‑CSF. The proportion of antibodies recognizing the 2G12 epitope was slightly increased, but the change was not statistically significant. These results indicated that the E

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METHODS

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ASSOCIATED CONTENT

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spectra, and the MALDI-TOF MS data of the compounds (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants (2012CB822100, 2013CB910700) from the Ministry of Science and Technology of China and the National Natural Science Foundation of China (21232002, 81473084).

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REFERENCES

(1) Horiya, S., MacPherson, I. S., and Krauss, I. J. (2014) Recent strategies targeting HIV glycans in vaccine design. Nat. Chem. Biol. 10, 990−999. (2) Back, N. K., Smit, L., De Jong, J. J., Keulen, W., Schutten, M., Goudsmit, J., and Tersmette, M. (1994) An N-glycan within the human immunodeficiency virus type 1 gp120 V3 loop affects virus neutralization. Virology 199, 431−438. (3) Bolmstedt, A., Sjolander, S., Hansen, J. E., Akerblom, L., Hemming, A., Hu, S. L., Morein, B., and Olofsson, S. (1996) Influence of N-linked glycans in V4-V5 region of human immunodeficiency virus type 1 glycoprotein gp160 on induction of a virus-neutralizing humoral response. J. Acquired Immune Defic. Syndr. Hum. Retrovirol. 12, 213− 220. (4) McCaffrey, R. A., Saunders, C., Hensel, M., and Stamatatos, L. (2004) N-linked glycosylation of the V3 loop and the immunologically silent face of gp120 protects human immunodeficiency virus type 1 SF162 from neutralization by anti-gp120 and anti-gp41 antibodies. J. Virol. 78, 3279−3295. (5) Binley, J. M., Ban, Y. E., Crooks, E. T., Eggink, D., Osawa, K., Schief, W. R., and Sanders, R. W. (2010) Role of complex carbohydrates in human immunodeficiency virus type 1 infection and resistance to antibody neutralization. J. Virol. 84, 5637−5655. (6) Grundner, C., Pancera, M., Kang, J. M., Koch, M., Sodroski, J., and Wyatt, R. (2004) Factors limiting the immunogenicity of HIV-1 gp120 envelope glycoproteins. Virology 330, 233−248. (7) Shan, M., Klasse, P. J., Banerjee, K., Dey, A. K., Iyer, S. P., Dionisio, R., Charles, D., Campbell-Gardener, L., Olson, W. C., Sanders, R. W., and Moore, J. P. (2007) HIV-1 gp120 mannoses induce immunosuppressive responses from dendritic cells. PLoS Pathog. 3, e169. (8) Huang, X., Jin, W., Hu, K., Luo, S., Du, T., Griffin, G. E., Shattock, R. J., and Hu, Q. (2012) Highly conserved HIV-1 gp120 glycans proximal to CD4-binding region affect viral infectivity and neutralizing antibody induction. Virology 423, 97−106. (9) Pollakis, G., Kang, S., Kliphuis, A., Chalaby, M. I., Goudsmit, J., and Paxton, W. A. (2001) N-linked glycosylation of the HIV type-1 gp120 envelope glycoprotein as a major determinant of CCR5 and CXCR4 coreceptor utilization. J. Biol. Chem. 276, 13433−13441. (10) Geijtenbeek, T. B., Kwon, D. S., Torensma, R., van Vliet, S. J., van Duijnhoven, G. C., Middel, J., Cornelissen, I. L., Nottet, H. S., KewalRamani, V. N., Littman, D. R., Figdor, C. G., and van Kooyk, Y. (2000) DC-SIGN, a dendritic cell-specific HIV-1-binding protein that enhances trans-infection of T cells. Cell 100, 587−597. (11) Trkola, A., Purtscher, M., Muster, T., Ballaun, C., Buchacher, A., Sullivan, N., Srinivasan, K., Sodroski, J., Moore, J. P., and Katinger, H. (1996) Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70, 1100−1108. (12) Scanlan, C. N., Pantophlet, R., Wormald, M. R., Saphire, E. O., Stanfield, R., Wilson, I. A., Katinger, H., Dwek, R. A., Rudd, P. M., and Burton, D. R. (2002) The broadly neutralizing anti-human

The detailed methods for the preparation of compounds, mice immunization, and serological assays were provided in the Supporting Information.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00224. The synthesis of carbohydrate building blocks, the preactivation-based one-pot synthesis of tetramannoside, the synthesis of triantennary pentamannoside, the deprotection of the oligomannoses, the method of protein conjugation, the method of mice immunization, the method of serological assays, NMR spectra, HRMS F

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DOI: 10.1021/acschembio.6b00224 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acschembio.6b00224 ACS Chem. Biol. XXXX, XXX, XXX−XXX