key: cord-0904976-6bzss27n
authors: Silva de Castro, Isabela; Gorini, Giacomo; Mason, Rosemarie; Gorman, Jason; Bissa, Massimiliano; Rahman, Mohammad A.; Arakelyan, Anush; Kalisz, Irene; Whitney, Stephen; Becerra-Flores, Manuel; Ni, Eric; Peachman, Kristina; Trinh, Hung V.; Read, Michael; Liu, Mei-Hue; Van Ryk, Donald; Paquin-Proulx, Dominic; Shubin, Zhanna; Tuyishime, Marina; Peele, Jennifer; Ahmadi, Mohammed S.; Verardi, Raffaello; Hill, Juliane; Beddall, Margaret; Nguyen, Richard; Stamos, James D.; Fujikawa, Dai; Min, Susie; Schifanella, Luca; Vaccari, Monica; Galli, Veronica; Doster, Melvin N.; Liyanage, Namal P.M.; Sarkis, Sarkis; Caccuri, Francesca; LaBranche, Celia; Montefiori, David C.; Tomaras, Georgia D.; Shen, Xiaoying; Rosati, Margherita; Felber, Barbara K.; Pavlakis, George N.; Venzon, David J.; Magnanelli, William; Breed, Matthew; Kramer, Josh; Keele, Brandon F.; Eller, Michael A.; Cicala, Claudia; Arthos, James; Ferrari, Guido; Margolis, Leonid; Robert-Guroff, Marjorie; Kwong, Peter D.; Roederer, Mario; Rao, Mangala; Cardozo, Timothy J.; Franchini, Genoveffa
title: Anti-V2 antibodies virus vulnerability revealed by envelope V1 deletion in HIV vaccine candidates
date: 2021-01-09
journal: iScience
DOI: 10.1016/j.isci.2021.102047
sha: 0644f0d1f32b7a3c04549c088c8d08249a519917
doc_id: 904976
cord_uid: 6bzss27n

The efficacy of ALVAC-based HIV and SIV vaccines in humans and macaques correlates with antibodies to envelope variable region 2 (V2). We show here that vaccine-induced antibodies to SIV variable region 1 (V1) inhibit anti-V2 antibody-mediated cytotoxicity and reverse their ability to block V2 peptide interaction with the α(4)β(7) integrin. SIV vaccines engineered to delete V1 and favor an α helix, rather than a β sheet V2 conformation, induced V2-specific ADCC correlating with decreased risk of SIV acquisition. Removal of V1 from the HIV-1 clade A/E A244 envelope resulted in decreased binding to antibodies recognizing V2 in the β sheet conformation. Thus, deletion of V1 in HIV envelope immunogens may improve antibody responses to V2 virus vulnerability sites and increase the efficacy of HIV vaccine candidates.

The HIV recombinant canarypox-derived vector (ALVAC) and gp120-envelope proteins formulated in alum vaccine platform tested in the RV144 HIV vaccine trial was the first to reduce the risk of HIV acquisition in humans (31.2%) (Rerks-Ngarm et al., 2009) . Serum IgG to the gp70-V1/V2 scaffold (Haynes et al., 2012) and to linear V2 peptides (Gottardo et al., 2013; Zolla-Pazner et al., 2014) have been identified as correlates of reduced risk of HIV acquisition. Sieve analysis further demonstrated genetic markers of immunologic pressure at positions 169 and 181 (Rolland et al., 2012) of V2, a region that binds to the a 4 b 7 integrin (Lertjuthaporn et al., 2018) . V2 is structurally polymorphic and can adopt b strand or a-helical conformations. However, V2 interaction with the a 4 b 7 integrin is inhibited preferentially by antibodies recognizing its a-helical conformation (Lertjuthaporn et al., 2018) .

The SIV mac251 macaque model, in which vaccinated animals are mucosally exposed to the highly pathogenic SIV mac251 at a dosage far in excess of HIV transmission in humans, recapitulated the modest vaccine efficacy observed in RV144 and identified antibodies to V2 as a correlate of reduced risk of SIV acquisition (Pegu et al., 2013; Vaccari et al., 2016) . Furthermore, substitution of the alum adjuvant with MF59 (Vaccari et al., 2016) in the same animal model abolished the vaccine protection afforded by the ALVAC/gp120 vaccine platform, thereby predicting the recently announced lack of efficacy in the HVTN-702 HIV trial that used the MF59 adjuvant (Cohen, 2020).

Antibodies to a V1 region adjacent to V2 (V1a) are associated with increased SIV mac251 acquisition

We investigated the serum antibody responses to V1 and V2 using linear peptide arrays in a cohort of 78 macaques immunized with four different vaccine regimens and exposed by the same route to the same dose of an identical SIV mac251 stock ( Figure S1A ). The gp120 protein bivalent boost was adjuvanted in alum in three regimens (ALVAC-SIV/gp120 + alum, DNA-SIV/ALVAC-SIV/gp120 + alum, and Ad26-SIV/AL-VAC-SIV/gp120 + alum) and in MF59 in the fourth (ALVAC-SIV/gp120 + MF59) (Vaccari et al., 2016 (Vaccari et al., , 2018 . The efficacy of these regimens was evaluated as the average per-challenge risk of SIV mac251 acquisition compared with unvaccinated controls following intrarectal exposure to repeated, low doses of the virus. For simplicity, we hereafter refer to ALVAC-SIV/gp120 + alum and the DNA-SIV/ALVAC-SIV/gp120 + alum (with respective vaccine efficacies of 44% and 52%; p < 0.05) as the protective regimens (Figures S1B and S1C), and to ALVAC-SIV/gp120 + MF59 and Ad26-SIV/ALVAC-SIV/gp120 + alum (vaccine efficacies of 9% and 13%; p > 0.05) as non-protective regimens (Figures S1D and S1E) (Vaccari et al., 2016 (Vaccari et al., , 2018 .

The levels of sera antibody reactivity to overlapping linear V1 (Starcich et al., 1986) peptides 15-24 ( Figure S1F ) did not differ between protective and non-protective vaccines ( Figure S1G ). However, when all vaccinated macaques were analyzed, animals with above-median antibody levels to V1 had a trend toward increased risk of SIV mac251 acquisition (p = 0.0658; Figure S1H ). Analysis of antibody responses to V1 peptides in protective and non-protective vaccine subgroups showed that anti-V1 antibodies were associated with an increased risk of SIV mac251 acquisition only in the non-protective group (Figures 1A and S1I) . Antibody responses to V1 peptides 23 and 24, encompassing the amino acid segment NETSSCIAQNNCTGLEQEQMISCKF, revealed higher reactivity in the non-protective vaccine subgroup when compared with the protective group ( Figure 1B) . The region designated here as V1a (peptide 23 to 24) lies directly N-terminal to a cryptic a 4 b 7 integrin-binding site (Tassaneetrithep et al., 2014) in V2 (V2b), with both V1a and V2b being part of a continuous, exposed peptide segment at the extreme apex of the envelope trimer (Gorman et al., 2016; Julien et al., 2013; Liu et al., 2008; Pancera et al., 2014) (Figures 1C and 1D) . V1a is also in tertiary contact with the V2 region (V2c) that contains the canonical tripeptide shown to bind to the a 4 b 7 integrin (Arthos et al., 2008; Nakamura et al., 2012) , as depicted in Figure 1D in a 3D homology model of the SIV mac251 trimer based on the HIV BG505 cryo-EM structure. The V1/V2 domain of a related cryo-EM structure of SIVcpzPtt is nearly identical (Andrabi et al., 2019) . Based on these structural relationships, we hypothesized that antibodies to V1a may influence vaccine efficacy by interfering with antibody binding to V2 and tested different assays of anti-V1 and anti-V2-specific monoclonal antibodies (mAbs) cloned from vaccinated protected or vaccinated SIV-infected animals (Mason et al., 2016) .

Antibody to V1a decreases anti-V2 antibody cytotoxicity and ability to inhibit gp120 binding to a 4 b 7

The mAbs (NCI09 and NCI05) cloned from the vaccinated and protected animal P770 (Vaccari et al., 2016) were cross-reactive with SIV mac251 and SIV smE543 gp120, V1/V2 scaffolds, and cyclic V2 peptides ( Figures  S2A-S2C ). Linear peptide mapping ( Figure S2D ), peptide competition ( Figures S2E and S2F) , and crystallography ( Figures S3 and S4 ; PDB: 6VRY) demonstrated that NCI09 recognized the TGLKRDKTKEY epitope in V2b. NCI05 did not bind to linear SIV mac251-K6W peptides ( Figure S2C ), but its binding to cyclic V2 was competed by peptides encompassing the SIV mac239 TGLKRDKKKEYNETWYSAD amino acid sequence ( Figure S2F ). From the same animal, we also obtained two V1-specific mAbs, NCI04 and NCI06, recognizing the CNKSETDRWGLTK epitope located N-terminal to V1a (Figures 1C and S2C) . None of these mAbs neutralized tier 2 SIV mac251 or SIV SME660. NCI05 neutralized tier 1 SIV SME660 but not tier 1 SIV mac251 . NCI06 and NCI09 had low neutralizing activity against tier 1 SIV mac251 ( Figure S2G ). Both NCI05 and NCI09 bound to gp120 on the surface of Gag-positive SIV mac239 -infected cells (Figures 1E, 1F, S5A, and S5B) and to SIV mac251 virions ( Figure 1G ). Functionally, NCI05 and NCI09 mAbs inhibited SIV gp120 binding to the a 4 b 7 integrin in a cell adhesion assay (Lertjuthaporn et al., 2018; Wibmer et al., 2018) (Figure 2A ) and mediated antibody-dependent cell-mediated cytotoxicity (ADCC; Figure 2B ). Of (B) Serum antibody against peptides 23 and 24 (V1a) in animals vaccinated with protective (diamonds, n = 39) and nonprotective (inverted triangles, n = 39) vaccines 3 weeks after the last immunization and 1 week before challenge (week 27), data shown as mean with SD. (C) Amino acid sequence of V1 and V2 (SIV mac251-K6W ). Sequences are represented as follows: V1 (black); V1a (black dotted line); V2 (red); V2b (red dotted line; recognized by mAb NCI09); V2c (green dotted line; recognized by mAb NCI05). MAb ITS41 recognizes the V1a epitope, and NCI04 and NCI06 recognize amino acids in the N-terminal region of V1. (D) Spatial relationship of V1 (olive), V2b (red), V2c (green), and the canonical V2 tripeptide, DLV, that binds the a 4 b 7 integrin (purple) in the gp120 trimer. (E) NCI05 and NCI09 binding to SIV mac239 -infected A66 p24 Gag-positive cells in a representative experiment of staining of SIV mac251 -infected cells. (F) The average percentage of Gag and NCI05 positive (green) or NCI09 positive (red, n = 2), data shown as mean with SD. (G) SIV mac251 virion capture assay (n = 7 experiments, black dots): virion input (gray) or virion captured by beads coated with NCI05 (green), NCI09 (red), or mouse IgG (negative control) (Rhesus IgG isotype was also used as a negative control, with n = 3, mean = 5.09 SIV RNA in transformed log copies/mL and SD = 0.08. Both NCI05 and NCI09 mAb were ll OPEN ACCESS iScience 24, 102047, February 19, 2021 3 iScience Article interest, the mAb ITS41 recognizing V1a (Mason et al., 2016) inhibited binding of NCI09 to the gp120 on the surface of SIV mac251 -infected cells ( Figure 2C ), as well as the binding of NCI05 and NCI09 mAbs to gp120 SIV mac251-M766 (Lertjuthaporn et al., 2018; Wibmer et al., 2018) (Figures 2D and 2E ). Prebound NCI05 and NCI09 were not affected by ITS41, demonstrating asymmetric competition (Figures S5C and S5D) . The NCI06 mAb, which recognizes a peptide distal to V1a not in contact with V2b or V2c in the 3D envelope structure ( Figures 1C and 1D ), did not interfere with NCI09 binding to gp120 (Figures S5E and S5F) . In addition, increasing amounts of ITS41 reversed NCI09 inhibition of gp120 binding to the a 4 b 7 integrin (Figures 2F and S5G) and inhibited NCI05 and NCI09-mediated ADCC (Figures 2G and 2H) . NCI04 did not affect NCI05 or NCI09-mediated ADCC (Figures 2I and 2J) . Of interest, both ITS41 and NCI04 mediate ADCC to a much lower extent than NCI05 and NCI09 despite having the identical Fc region since all antibodies were cloned in an expression vector that joined variable regions with the same Fc scaffold. This result highlights the importance of Fab properties and epitope accessibility for ADCC.

V1-deleted immunogens designed to favor V2 a-helical conformation reduce the risk of SIV mac251 acquisition

The functional interference of ITS41 with NCI05 and NCI09 raised the hypothesis that deletion of V1 in SIV/ HIV envelope immunogens could increase V2 accessibility, enhance the level of V2 functional antibodies, and increase vaccine efficacy. To test this, we designed V1-deleted gp120 proteins (gp120 DV1 ) by symmetrically truncating V1 at its stem, since its origin and insertion (stem) to the V1/V2 domain connect the A and B b strands (McLellan et al., 2011) . The V1/V2 domain remaining after deletion of the gp120 V1 (gp120 DV1 ) was energy minimized as previously described Cardozo et al., 1995) . The search predicted a stable, low-energy, partially a-helical V2 conformation in gp120 ( Figure 3A and Table S1 ). As control, we designed another V1-deleted gp120 (gp120 DV1gpg ) by inserting the Gly-Pro-Gly b turn at the excision point with the purpose of minimizing disruption to the crystallographically visualized V1/V2

Greek key b sheet fold (Figures 3B and Table S2 ). We then expressed M766-based gp120 DV1 and gp120 DV1gpg proteins in Chinese Hamster Ovary (CHO) cells together with wild-type gp120 (gp120 WT ; Table S3 ). The purified monomeric gp120 DV1 and gp120 DV1gpg proteins were stable and unrecognized by the anti-V1 NCI06 and ITS41 mAbs ( Figure S5H ), bound to NCI05 and NCI09 by ELISA ( Figures 3C-3E ), immune precipitation, and western blot better than gp120 WT (Figures S5H and S5I) . Of interest, gp120 DV1 and gp120 DV1gpg also bound better to simian soluble CD4 than the gp120 WT ( Figure 3F ). These data are consistent with increased exposure of V2 epitopes and the CD4-binding site in the gp120 DV1 and gp120 DV1gpg antigens (Ching and Stamatatos, 2010).

We tested the efficacy of the V1-deleted immunogens using a DNA-SIV-prime/ALVAC-SIV/with gp120 protein + alum monovalent boost regimen followed by low-dose intrarectal exposures to SIV mac251 . We designed a modified vaccine regimen aimed at magnifying a possible difference in the efficacy of the wild type and DV1 immunogens. Here, we halved the amount of SIV Gag DNA in the prime and performed a single protein boost (rather than two) with ALVAC-SIV using the SIV mac251 gp120 M766 alone, omitting the two SIV SME543 gp120 GC7V protein boosts (Vaccari et al., 2016 (Vaccari et al., , 2018 . In previous studies, the association between antibodies to V2 and a decreased risk of SIV mac251 acquisition were notably revealed by SIV SME543 antigens but, curiously, not by SIV mac251 antigens.

We vaccinated three groups of 14 macaques each with two inoculations (weeks 0 and 4) of plasmid DNAs expressing SIV gp160 WT (group 1; Table S4 ), SIV gp160 DV1 (group 2; Table S5 ), or SIV gp160 DV1gpg (group 3; Table S6 ) together with SIV p57 Gag. All groups received one boost at week 8 with ALVAC-SIV expressing gp120 WT , and each group was administered a final boost at week 12 consisting of the same AL-VAC-SIV together with the SIV mac251-M766 gp120 WT (group 1), gp120 DV1 (group 2), or gp120 DV1gpg (group 3) protein adjuvanted with alum Alhydrogel ( Figure 3G ). Alongside a simultaneous control group of 18 naive macaques, the vaccinated macaques were exposed weekly to a total of 11 low doses of SIV mac251 by the intrarectal route, beginning at 5 weeks from the last immunization (week 17). A significant Figure 3 . Vaccine design and virological outcome (A) Model of the gp120 DV1 V2 a helix (green). The predicted a-helical structure of the V2c peptide was imposed on the V2c segment in gp120 and the local backbone energy minimized.

(B) Model of the gp120 DV1gpg b strand.

(C-E) ELISA binding of the purified (C) gp120 WT , (D), gp120 DV1 , and (E) gp120 DV1gpg with a-V2 mAbs NCI05 and NCI09 and the a-V1 mAb NCI06.

(F) Binding of simian CD4-Ig to gp120 WT , gp120 DV1 , and gp120 DV1gpg . (G) Schematic representation of the study design. Each vaccinated group included 14 young macaques, and the control group consisted of 18 naive young macaques. All animals were simultaneously exposed to weekly low doses of SIV mac251 by the intrarectal route beginning at week 17. iScience Article decrease in the risk of SIV mac251 acquisition was observed following immunization with the gp160 DV1 DNA and gp120 DV1 protein immunogens engineered predominantly to favor the a-helical V2 conformation (vaccine efficacy 57%; p = 0.04; Figure 3H ) but not following vaccination with wild-type envelope immunogens in group 1 or DV1gpg envelope immunogens in group 3 ( Figures S6A and S6B ). We observed no sustained, significant difference in the level of plasma viral RNA in animals that became infected in each vaccinated group compared with controls ( Figures S6C-S6I ) and only a transient trend of decreased SIV DNA levels in the rectal mucosa in group 2 ( Figure S6J ). The lack of vaccine efficacy in group 1 was not entirely unexpected given both the decreased amount of DNA used in the prime and, perhaps more importantly, the omission of the two SIV SME660 gp120 GC7V boosts, as the antibody level to cyclic V2 E543 was a main correlate of reduced risk in two independent studies (Vaccari et al., 2016 (Vaccari et al., , 2018 . The different outcomes in groups 2 and 3 compared with controls suggested that the inferred differences in the V2 conformation of the DV1 and DV1gpg immunogens might have quantitatively or qualitatively affected the antibody response to V2.

Antibodies inhibiting V2-a 4 b 7 interaction are associated with a decreased risk of SIV mac251 acquisition Protection in RV144 correlated with a non-glycosylated V2 peptide that adopts an a-helical 3D conformation (Aiyegbo et al., 2017) and encompasses the canonical a 4 b 7 integrin-binding site in V2. In previous macaque studies, Ab binding to the V2 SIV SME543 peptide (but not SIV mac251 ) was associated with a decreased risk of SIV mac251 acquisition (Vaccari et al., 2016 (Vaccari et al., , 2018 . We therefore engineered an isolated V2 peptide (V2c E543 ) corresponding to the V2c region of SIV SME543 (a clone of SIV SME660 ) by identifying the beginning and ending amino acids between V2 positions 165 and 181 that adopt an a-helical conformation. Serum reactivity to the V2c E543 peptide (DKKIEYNETWYSRD) was higher (trend) in animals immunized with the DV1 immunogens ( Figure 3I ) and correlated with a decreased risk of SIV mac251 acquisition (R = 0.36, p = 0.02; Figure 3J) . Furthermore, the level of inhibition of V2c E543 binding to the a 4 b 7 integrin in the eight animals that had values above the cutoff of the assay correlated with a decreased risk of SIV mac251 acquisition (R = 0.73, p = 0.046; Figures 3K and S7A) . Reactivity to an equivalent SIV mac251 peptide did not reveal any association with risk of acquisition (data not shown). These animals were all immunized with SIV mac251based immunogens, suggesting that the V2c E543 conformation is better able to capture antibodies associated with a decreased risk of SIV mac251 acquisition, in agreement with prior observations (Vaccari et al., 2016 (Vaccari et al., , 2018 . Serum recognition of SIV mac251 V2 linear (Figures S7B-S7D) or cyclic peptide ( Figure S7E ), of the entire gp120 peptide array (sorted as responses to V4, C3, and C5, with responses to C3 and C5 being highest in animals immunized with the DV1 immunogens; Figures S7F-S7H), had no apparent association with a decreased risk of SIV mac251 acquisition.

ADCC to gp120-coated cells or SIV-infected cells correlates with a decreased risk of SIV mac251 acquisition ADCC was a secondary correlate of reduced risk in individuals with low IgA levels in RV144 (Tomaras et al., 2013) . ADCC activity mediated by the plasma from animals in the WT, DV1, and DV1gpg groups was performed using the target EGFP-CEM-NKr-CCR5-SNAP cells (Orlandi et al., 2016 ) (T lymphoblastoid cell line CEM-based assay) coated with purified gp120 WT , gp120 DV1 . Analysis of the coated cells demonstrated that NCI05 or NCI09 bound less well to CEM cells coated with gp120 WT than those coated with gp120 DV1 and gp120 DV1gpg (Figures S8A-S8D ). However, ADCC measured with cells coated with the gp120 WT did not differ among the animal groups ( Figures 4A-4C ), suggesting that V1 is not a major target of ADCC. Animals vaccinated with the DV1 immunogen (group 2) mounted significantly higher ADCC titers directed to DV1 than to the DV1gpg antigen ( Figure 4B ). Animals immunized with the DV1gpg immunogen (group 3) mounted lower ADCC titers directed to DV1gpg than to DV1 antigens (Figures 4C and Table S7 ). ADCC . Continued (J) Correlation between serum antibodies to V2c E543 and number of intrarectal challenges (WT, n = 14; DV1, n = 14; DV1gpg, n = 13).

(K) Correlation of serum inhibition of the a 4 b 7 integrin (expressed on RPMI8866 cells) to V2c E543 and intrarectal challenges (WT, n = 2; DV1, n = 2; DV1gpg, n = 4). The correlation was performed only with data from animals that had inhibition above the assay cutoff. The infection curves were analyzed using Log Rank (Mantel-Cox test); data comparison between the three vaccinated groups was done with non-parametrical Kruskal-Wallis test with Dunn's multiple comparison test. The correlation analyses were performed using the non-parametric Spearman rank correlation method with the exact permutation two-tailed p-values calculated. See Tables S1-S6 and Figures S5-S7. ll

iScience 24, 102047, February 19, 2021 7 iScience Article Figure 4 . ADCC directed to DV1 gp120 associated with decreased risk of SIV mac251 acquisition (A-C) CEM-based ADCC titers in animals immunized with (A) WT, (B) DV1, or (C) DV1gpg envelope immunogens on target cells coated with gp120 WT , gp120 DV1 , or gp120 DV1gpg (WT, n = 14; DV1, n = 14; DV1gpg, n = 13 animals). Data shown as mean with SD.

(D-F) Correlation of ADCC titers directed to the DV1 antigen in animals immunized with (D) WT, (E) DV1, or (F) DV1gpg envelope immunogens and time of SIV mac251 acquisition (WT, n = 14; DV1, n = 14; DV1gpg, n = 13 animals).

(G and H) Correlation of ADCC titers in animals immunized with (G) DV1gpg or (H) WT envelope immunogens on target cells coated with gp120 WT and time of SIV mac251 acquisition (WT, n = 14; DV1gpg, n = 13 animals). iScience Article was also performed using target cells infected with SIV mac251 (Lewis et al., 2019) , and no differences were observed among the animal groups in this assay ( Figure S8E ). These data demonstrate that the two V1deleted immunogens differ both in their ability to induce and to reveal cytotoxicity activity when used in the CEM-based ADCC assay.

We performed correlation analyses using the non-parametric Spearman test to assess the relationship of ADCC titers to the three antigens and the risk of SIV mac251 acquisition and found that ADCC directed to gp120 DV1 protein is significantly correlated with a reduced acquisition risk in the WT and DV1-vaccinated groups, and a correlation ( Table S7 ), indicative of the DV1 antigen's ability to capture protective antibodies. In group 3, a correlation with decreased acquisition was also observed with ADCC directed to WT gp120 (R = 0. 61, p = 0.03; Figures 4G and Table S7 ). Strikingly, there was a correlation with ADCC directed to WT and an increased risk of SIV mac251 acquisition in animals immunized with the WT immunogens (R = À0.55; p = 0.04; Figures 4H and Table S7 ), suggesting that ADCC leading to a decreased risk of viral infection in all groups is better elicited and exhibited in the CEM-based ADCC assay by the DV1 protein.

ADCC activity measured on SIV mac251 -infected cells demonstrated no difference among the vaccinated groups ( Figure S8E ). However, the percentage of specific ADCC killing of SIV mac251 -infected cells correlated significantly with a decreased risk of SIV mac251 acquisition in the non-parametric Spearman test only in the DV1-immunized group (R = 0.58, p = 0.03; Figure 4I ). In this group, the level of ADCC directed to CEM cells coated with the DV1 protein correlated (trend) with the ADCC measured against infected cells (R = 0.43, p = 0.13; Figure 4J ) and the frequency of vaccine-induced T helper (Th) 2 cells (R = 0.72, p = 0.007; Figures 4K, S8F, and S8G), suggesting that Th2 cells promote protective ADCC activity.

V2-specific ADCC, but not neutralizing antibody titers, correlates with a decreased risk of SIV mac251 acquisition Next, we examined the contribution of anti-V2 antibodies to the ADCC measured with the CEM-based assay by using purified NCI05 and NCI09 F(ab') 2 as competitor, since both antibodies proved equally capable of mediating ADCC against gp120 DV1 -coated cells (Figures 2A and 2B ). Both the NCI05 and NCI09 F(ab') 2 competed approximately 40% of serum ADCC directed to the gp120 DV1 antigen in animals immunized with this immunogen (group 2; Figures 5A and 5B and Figures S8H-S8K ). Of importance, the V2specific serum ADCC activity inhibited by NCI05 or NCI09 F(ab') 2 in the DV1-immunized group (NCI05 mean delta = 15.12% +/À 2.96; NCI09 = 14.09% +/À 5.21) correlated with a decreased risk of SIV mac251 acquisition (NCI05, R = 0.67, p = 0.01; NCI09, R = 0.54, p = 0.05; Figures 5C and 5D ), whereas the remaining non-V2-specific ADCC activity did not (data not shown).

An identical analysis of the sera of animals immunized with the DV1gpg immunogen demonstrated approximately 20% inhibition by both mAbs F(ab') (NCI05 mean delta = 8.60% +/À 3.05; NCI09 = 8.74% +/À 3.18; Figures 5E , 5F, S8L, and S8M). We observed no correlation with V2-specific ADCC activity inhibited by NCI05 or NCI09 (delta) and the risk of SIV mac251 acquisition in this group ( Figures 5G and 5H ). The level of estimated V2-specific serum ADCC activity inhibited by either NCI05 or NCI09 F(ab') 2 was significantly higher in the DV1 than the DV1gpg group (NCI05: p = 0.0001; NCI09: p = 0.0040; Figures 5I and 5J ). Extension of our analyses to antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent neutrophil activation (ADNP) using beads coated with the gp120 DV1 protein (Mahan et al., 2016) revealed that none of these responses correlated with SIV mac251 acquisition ( Figures S8N and S8O and data not shown). Serum neutralizing antibody titers against the tier 2 SIV mac251-CS41 were highest in the DV1 group as expected (Ching and Stamatatos, 2010) ( Figure S8P ) but did not correlate with the risk of infection (data not shown). Neutralizing titers to tier 1B SIV mac251-M766 did not differ among the immunization groups (Figure S8Q ), but strikingly, their levels correlate with an increased risk of SIV mac251 acquisition in the DV1 group iScience Article (Table S8 and Figure S8R ). These data support the idea that the DV1 immunogen engineered to favor a V2 a-helical conformation to elicit qualitatively different V2-specific ADCC titers providing a plausible explanation for the difference in vaccine efficacy observed in groups 2 and 3.

We tested whether the cytocidal function of V2-specific antibody limits the early seeding of the virus in vaccinated animals that become infected. First, we measured serum reactivity to linear V2 peptides 27 and 29 (corresponding to V2b and V2c) in animals immunized with protective vaccines, including group 2 described in the current study (Figures 3H, S1B, and S1C), or with non-protective vaccines, including groups 1 and 3 of the current study ( Figures S6A, S6B , S1D, and S1E). An inverse correlation with serum reactivity and the level of SIV DNA in the rectal mucosa was found in protective ( Figures 5M and 5N ). Collectively, these data suggest that anti-V2 antibodies may inhibit infection by more than one mechanism.

V1 deletion in HIV A244 gp120 decreases V2 b sheet conformation

To address the potential for differences in HIV and SIV envelope structures, we tested the relevance of our finding to HIV by generating two V1-deleted HIV clade A/E A244 gp120 proteins. The first, A244DV1 a , was designed by deleting the LTNVNNRTNVSNIIGNITD peptide and leaving the natural nine-amino-acid loop in V1 (Table S10 and Figure S9A ) with the intent of minimizing tension in the adjacent V2 loop. In the second construct, A244DV1 b , the nine-amino-acid loop was replaced with the corresponding nine amino acids of the SIV mac251 DV1 antigen already proven to favor an a-helical V2 structure eliciting cytocidal antibodies correlating with a decreased risk of SIV acquisition (Table S11 and Figure S9A ). A244WT, A244DV1 a , and A244DV1 b (Tables S9-S11) gp120 antigens expressed in 293 cells were probed in ELISA with mAb PG9, which recognizes V2 in a b sheet conformation, or CH58 and CH59, which recognize V2c in an a-helical conformation (Bonsignori et al., 2012; Gorny et al., 1994; Liao et al., 2013) . The PG9 mAb bound better to gp120 A244 WT than to both the gp120 A244 DV1a and gp120 A244 DV1b proteins ( Figures 6A and 6B ). The CH58 and CH59 mAbs had similar reactivity to A244 WT and the A244 DV1a and A244 DV1b proteins , suggesting that V1 deletion in the HIV gp120 A244DV1 a and gp120 A244DV1 b proteins shifts the structural equilibrium of V2 and reduces the V2 b sheet conformation (recognized by PG9) without affecting the V2 a-helical conformation (recognized by CH58 and CH59). These DV1 HIV immunogens therefore represent reagents matched to the V1-deleted SIV immunogens (that reduced the risk of virus acquisition) suitable to test whether focusing the antibody response to a V2 a-helical conformation improves the efficacy of HIV vaccine candidates.

We have herein demonstrated that antibodies to the V1 region, exposed at the apex of the virion envelope trimer and adjacent to a conserved V2 region, have opposing effects on SIV mac251 acquisition in vaccinated macaques. Anti-V2 antibodies bound to infected cells and virions inhibited V2 binding to a 4 b 7 and mediated ADCC, whose level and function correlated with a decreased risk of virus acquisition. In contrast, anti-V1 antibodies interfered with the ability of anti-V2 antibodies to bind gp120, mediate ADCC, and inhibit gp120 and a 4 b 7 integrin interaction and furthermore correlated with an increased risk of virus acquisition. Whether antibodies to V1 affect V2 recognition by rendering specific V2 Ab-targeted epitopes less accessible by steric hindrance (competitive) or by stabilizing V1/V2 in a Greek-key b sheet fold through allosteric (non-competitive) inhibition remains to be determined. These mechanisms immunized animals in the current study) that became infected (n = 60). Data represented as mean with SD. Data comparisons between two paired or unpaired groups were done with Wilcoxon signed-rank test and Mann-Whitney test, respectively. The correlation analyses were performed using the non-parametric Spearman rank correlation method with exact permutation two-tailed p-values calculated. See Table S8 and Figures S1A-S1D, S6A-S6B, S7, and S8.

iScience 24, 102047, February 19, 2021 11 iScience Article Figure 6 . HIV A244DV1 gp120 immunogens are preferentially recognized by human antibodies binding to V2 in a-helix conformation (A, C and E) ELISA kinetic of HIV gp120 A244 WT, gp120 DV1 a , and gp120 DV1 b reactivity to (A) PG9 (anti-V2 antibody that recognizes V2 b Barrel conformation), (C) CH58, and (E) CH59 (anti-V2 antibodies recognizing V2 a-helical conformation). iScience Article are not mutually exclusive in the context of polyclonal antibody responses. We demonstrate here that vaccination with the DV1 immunogens engineered to favor the V2 a-helical conformation ( Figure 6G , center) elicited higher cytocidal V2 antibodies correlating with vaccine efficacy than immunogens with V2 in a b sheet conformation ( Figure 6G , right) that afforded no vaccine efficacy. Overall, the results presented here are consistent with the finding of an alternative, unconstrained V2 a-helical conformation, distinct from that visualized in most envelope crystallographic structures, that was targeted by antibodies from the sera of volunteers in RV144 (Aiyegbo et al., 2017) . This raises the hypothesis that V1 may not only decrease V2 accessibility to the immune system by direct masking but may also enforce a constrained V2 b strand conformation less accessible to the antibodies mediating ADCC via a non-competitive mechanism. Indeed, we demonstrate here that the SIV DV1 and DV1gpg antigens differed in their epitope accessibility. It is notable that the structure adopted by the isolated peptide V2c used in our studies differs significantly from the b strand form found in both the HIV-1 prefusion-closed trimer derived from stabilized gp120-gp41 linked by artificial disulfide bond (SOS) in combination with isoleucine-to-proline (IP) change in the gp41 (SOSIP) trimers (Medina-Ramírez et al., 2017) and the scaffolded V2 structures bound to HIV-1 bNAbs (Jiang et al., 2016) . The structure inferred in our study is instead closer to that identified in co-crystals of HIV V2 peptides in complex with mAbs derived from an uninfected RV144 vaccinee (Liao et al., 2013) , suggesting that the SIV V2 can adopt an a-helical structure analogous to the structure of V2 in HIV that has previously been linked to a reduced risk of HIV acquisition in humans (Lertjuthaporn et al., 2018) .

The V2 envelope region is important in viral transmission and seeding gut inductive sites, as inferred by studies on transmitted HIV variants (Cavrois et al., 2014; Chohan et al., 2005; Jiang et al., 2016; Ritola et al., 2004; Rong et al., 2007; Sagar et al., 2006; Smith et al., 2016) . Our findings here suggest that antibodies to V2 may interfere at different steps of viral transmission, from disrupting the interaction of the a-helical conformation of V2 with a 4 b 7 during the establishment of infection to inhibiting virus spread by ADCC. Indeed, a recent study by Goes et al., 2020 demonstrated that V2 interaction with a 4 b 7 provides a co-stimulatory signal that increases activation and proliferation of CD4 + cells and consequent HIV replication, suggesting that V2 may make gut resident T cells more receptive for viral infection. Furthermore, this phenotype was inhibited by antibodies recognizing the HIV a helix but not the b sheet conformation. Similar results were also obtained for SIV using the NCI09 mAb that was instrumental in our studies to reveal V2-specific ADCC responses correlating with a decreased risk of SIV mac251 acquisition ( Figure 5D ).

It is likely that the V1 of gp120 has evolved in SIV and HIV to counteract antibodies targeting the vulnerable V2 a-helical conformation. Antibody interference to HIV gp41 has been observed (Verrier et al., 2001) , but interfering antibodies that target the apical gp120 domains and the V1/V2 have not been previously described. Interfering antibodies have been described in other viruses as well, including the Western equine encephalitis, polio, hepatitis C, and influenza viruses and the SARS-coronavirus (Dulbecco et al., 1956; Nicasio et al., 2012; Sautto et al., 2012; To et al., 2012; Tripp et al., 2005; Zhong et al., 2009 ).

The V1 deletion strategy employed here is relevant to the HIV vaccine design as removal of V1 from the A244 gp120 envelope decreases mAb PG9 binding to the protein, as we have demonstrated. Of particular significance is that this detrimental V1 element remains a component of most current Env-based vaccine candidates, suggesting that these candidates may exhibit improved efficacy with V1 deletion.

In summary, we have demonstrated that antibodies to V1 counteract functional antibody responses to viral vulnerability sites in V2. Minimizing the confounding role of V1, by its deletion or other means, presents a new opportunity to understand the biochemical basis of V2-associated viral vulnerability toward developing a fully efficacious vaccine for HIV.

The current study demonstrates that SIV envelope immunogens with the V1 region deleted (DV1) to favor the V2 a-helical conformation induced significant vaccine protection, whereas deletion of V1 to favor the V2 b sheet conformation, or V1 repleted (Wild Type) SIV immunogens, were not protective. Although the DV1 immunogens were engineered to assume these different conformations, we were unable to confirm their conformation experimentally. It was encouraging, however, that V1 deletion in the HIV A244 envelope, iScience Article engineered to favor the V2 a-helical conformation, resulted in the loss of binding to monoclonal antibodies recognizing V2 in a b sheet conformation. In addition, the level and interference activity of V1specific antibodies may vary depending on the HIV clade (Shen et al., 2015) . Lastly, the predictive value of our preclinical study in Indian rhesus macaques for humans remains unclear. It is noteworthy, however, that the identical SIV mac251 macaque model recapitulated and predicted (Vaccari et al., 2016) the efficacy of the RV144 human trial (performed in more than 16,000 volunteers in Thailand) and the lack of efficacy of the HVTN702 trial (more than 5,000 volunteers in South Africa), respectively.

Further information and requests for resources and reagents should be directed to and will be fulfilled by Dr. Genoveffa Franchini (franchig@mail.nih.gov).

All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

The accession number for the structural analysis of the NCI09 antibody reported in this paper is PDB: 6VRY. All other data can be made available upon request.

All methods can be found in the accompanying Transparent methods supplemental file.

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.102047.

We thank D. Ahern for editorial assistance, K. Saunders for providing the vector for antibody expression, and J. Heeney, G. Alter, and S. Phogat for helpful discussion. We also thank Sanofi Pasteur for the AL-VAC-SIV vaccine. Funding: This work was supported with federal funds from the National Cancer Institute Intramural Program and the Office of AIDS research, National Institutes of Health (to G.F.) and under con- (3 weeks af ter the last immunization 1 week before challenge). Statistical analyses comparing two groups was done using Mann-Whitney test. The inf ection curves were analyzed using Log-Rank (Mantel-Cox test). with the gp120 immunogens and found that both mAbs stained a significantly lower percentage of cells coated with gp120WT than gp120∆V1 and gp120∆V1gpg, consistent with the improved binding of V1-deleted immunogens to CD4 shown in Figure 1F . Similarly, the Mean Fluorescent Intensity was also significantly lower in cells coated with WT than with V1-deleted immunogens following staining with ( Data represented as mean with SD. Data Comparisons between two paired or unpaired groups were perf ormed using Wilcoxon signed-rank test or Mann-Whitney test, respectively. Comparisons between the three vaccinated groups was done with non-parametrical Kruskal-Wallis test with Dunn's multiple comparison test. Comparisons between three paired groups was done with two-way ANOVA test with Tukey's multiple comparisons test. The correlation analyses were performed using the non-parametric Spearman rank correlation method with exact permutation two-tailed P values calculated.

NCI05 NCI09 NCI04 NCI06 ITS09 ITS41 SIVmac251 M766 gp120 ++ ++ ++ ++ ++ ++ SIVsmE660.CR54 gp140 ++ ++ - ++ ++ - SIVsmE543 1J08 V1V2 ++ ++ - ++ ND ND SIVmac251 1J08 V1V2 ++ ++ ++ ++ ND ND SIVmac239 1J08 V1V2 ++ ++ + ++ ND ND SIVsmE543 cV2 ++ ++ - - ND ND SIVmac251 cV2~ ¶ ++ - - ND ND V1 peptide - - PLCITMRCNKSETDRWGLTK RCNKSETDRWGLTK ND ND V2 peptide - TGLKRDKTKEY - - TGLKRDKKKEY EQEQMISCKFNMTGL ++ OD450 ≥ 2 + 1 ≤ OD450 < 2 - OD450 < 0.5 ¶ borderline 0.5 ≤ OD450

α-HIV gp120 Tables   Table S1 . DNA and protein sequence of SIV gp120ΔV1, Related to Figure 3 . The tPA signal peptide that is cleaved in the mature protein (blue) and the position where the V1 region has been deleted in the immunogen (orange) are shown. Table S2 . DNA and protein sequence of SIVgp120ΔV1gpg, Related to Figure 3 . The tPA signal peptide that is cleaved in the mature protein (blue) and the GPG sequence inserted in the immunogen (orange) are

shown. Table S3 . DNA and protein sequence of SIVgp120WT, Related to Figure 3 . The HSV-1 gD leader sequence that is cleaved in the mature protein (red) and the V1 region deleted in the ΔV1 immunogen (orange) are shown. Table S4 . DNA and protein sequence of SIVgp160WT, Related to Figure 3 . The V1 region deleted in the ΔV1 immunogen is shown in orange. Table S5 . DNA and protein sequence of SIVgp160ΔV1, Related to Figure 3 . The position where the V1 region has been deleted in the immunogen is shown in orange. Table S6 . DNA and protein sequence of SIVgp160ΔV1gpg, Related to Figure 3 . The GPG sequence inserted in the immunogen is shown in orange. Table S7 . ADCC titers and risk of SIVmac251 acquisition in the immunized groups, Related to Figure 4 .

Summary of ADCC activity measured in the immunized groups using the WT, ΔV1, and ΔV1gpg antigens to coat CEM cells. Table S8 . Serum neutralizing activity against tier 1, 2, and 3 SIV, Related to Figure 5 and Figure S8 . The data represented are serum neutralizing activity of samples collected at week 17 subtracted from neutralizing activity of samples collected at baseline. Table S9 . DNA and protein sequence of HIV AE.A244 D11gp120WT, Related to Figure 6 . The V1 stem sequence is shown in blue and the V1 region is shown in orange.

Table S10. DNA and protein sequence of HIV AE.A244 D11gp120 ∆V1a, Related to Figure 6 . Deletion of V1 is represented by brackets. Table S11 . DNA and protein sequence of HIV AE.A244 D11gp120 ∆V1b, Related to Figure 6 . Deletion of V1 is represented by brackets.

All animals used in this study were colony-bred rhesus macaques ( 131, the animal was challenged weekly for 10 weeks using 120 TCID50 of the same SIVmac251 challenge stock used at week 28 (Fig. S2) . Animal P770 remained uninfected.

The second cohort included three groups of 14 animals each, vaccinated intramuscularly with 1 mg of SIVp57Gag DNA together with 2 mg of either SIV gp160WT, ∆V1, or ∆V1gpg DNA at weeks 0 and 4. All animals were administered ALVAC-SIV alone at week 8, and ALVAC-SIV simultaneously with a gp120+alum SIVmac251 gp120M766 monovalent boost at week 12. Of note, a prior vaccine regimen using two inoculations of SIV-DNA (2 mg each of gp160 env and gag) and two of ALVAC-SIV administered simultaneously with a bivalent gp120+alum boosts SIVmac251 gp120M766 and SIVSM E660 gp120GC7V proteins) demonstrated 52% efficacy in macaques (Vaccari et al., 2018) . Curiously, the decreased risk of SIVmac251 acquisition in that study correlated not with the homologous cyclic V2 peptide from SIVmac251, but rather with the heterologous V2E543 f rom the SIVSME660 strain, suggesting that the V2 conformation may be more important than the primary amino acid sequence. Briefly, the DNA doses were f ormulated in sterile PBS in a f inal volume of 1.4mL containing 1 mg of SIVp57Gag DNA together with 2 mg of either SIV gp160WT, ∆V1, or ∆V1gpg, or SIV gp160 wild type DNA. Half of the dose was injected in each thigh, intramuscularly.

At weeks 8 and 12, all animals received an intramuscular immunization of 10 8 pfu of ALVAC-SIV vCP2432. At week 12, they received SIV M766 gp120WT, ∆V1, or ∆V1gpg protein formulated in alum and administered in the contralateral thigh. Beginning five weeks after the last immunization (week 17), all vaccinated animals and a group of 18 naïve control animals, were exposed intrarectally to one weekly dose of SIVmac251 (stock day 8 from 2010) TCID50 400 (calculated in Rhesus 221 cells). Viral load was evaluated weekly and animals testing negative for SIV RNA in plasma were re-exposed, for a maximum of 11 weekly challenges. Proteins were purified from the conditioned cell culture supernatant using a lectin-affinity chromatography (Galanthus nivalis lectin agarose; Vector Labs, Inc.) capture step, followed by anion exchange chromatography (Q-Sepharose; GE Healthcare Life Sciences) operated in flow through mode. Proteins were buffer exchanged into Dulbecco's phosphate buffered saline (DPBS) and filtered with 0.22µm filter.

The protein scaffold 1J08 has previously been shown to exhibit the SIV Env V1/V2 domain in the conf ormation naturally found on the native V1/V2 protomer basing on stable expression, clash score, and solvent accessibility. It was used here to identify V1/V2-specific B-cell clones and produced as previously Flow cytometric data was analyzed with FlowJo 9.7.5.

Total RNA was reverse-transcribed in each well, and the rhesus immunoglobulin heavy (H), light kappa (Lκ), and light lambda (Lλ) chain variable domain genes were amplified by nested PCR. Positive amplification products as analyzed on 2% agarose gel (Embi-Tec) were sequenced, and those that were identified as carrying either Igγ, IgLκ, or IgLλ sequences were re-amplified with sequence-specific primers carrying unique restriction sites using the first-round nested PCR products as a template. Resulting PCR products were run on a 1% agarose gel, purified with QIAGEN Gel Extraction Kit (QIAGEN), and eluted with 25µl of nuclease-free water (Quality Biological). Purified PCR products were then digested and 

The ability of mAbs to bind to SIVmac239 envelope expressed on the surface of infected A66 cells was evaluated by indirect surface staining using methods similar to those previously described 51 . Briefly 

Virions from SIV mac251 preparation were captured with 15 nm magnetic nanoparticles (MNPs) coupled to NCI05, NC09, mouse IgG, or Rhesus recombinant IgG1 (NHP Reagent Resource, clone DSPR1) mAbs as previously described (Arakelyan et al., 2013) . Briefly, carboxyl-terminated iron oxide nanoparticles (Ocean Nanotech, San Diego) were coated with 1 mg of mAbs according to manufacturer's protocol via two step carbodiimide reaction. In order to capture virions, MNPs coated with mAbs (3.9 x10 12 ) in 60 μl

were incubated with 100µl of viral preparation (10 ng/ml based on p27 content) for 1 h at 37°C. Captured virions were separated on MACS magnetic columns attached to an OctoMacs magnet (Miltenyi Biotech, Bergisch Gladbach, Germany), washed 4 times with 600 μl (0.5% bovine serum albumin, 2mM EDTA in PBS), and eluted in 100 μl PBS. The SIV RNA levels were measured by droplet digital PCR (ddPCR).

The ITS41 mAbs was isolated from an SIVsmE660-inf ected rhesus macaque. ITS41 recognizes the EQEQMISCKFTNMTGL peptide (sequence based on SIVmac239) that is part of the V1 epitope as previously reported (Mason et al.) . The monoclonal antibodies, NCI04, NCI06, NCI05, and NCI09 were generated in the present work. Binding of SIV-specific mAbs to viral proteins or synthetic peptides was measured by enzyme-linked immunosorbent assay (ELISA). Plates were coated overnight at 4ºC with 50 µl, 100 ng/well of antigen in PBS, then blocked with 300 µl/well of 1% PBS-BSA for 1 h at 37ºC. When cyclic V2 (cV2) was tested, plates were coated at 4ºC overnight with 200 ng/well of streptavidin (Sigma-Aldrich) in bicarbonate buffer, pH 9.6, then incubated with biotinylated cV2 peptide (produced by JPT Peptide Technologies) for 1 h at 37ºC and blocked with 0.5% milk in 1× PBS, 0.1% Tween 20, pH 7.4, overnight at 4ºC. Coated, blocked plates were incubated with 40 µl/well of serial dilutions of mAbs in 1% PBS-BSA for 1 h at 37ºC. Then, 40 µl/well of a polyclonal preparation of Horseradish peroxidase conjugated goat anti-monkey IgG antibody (Abcam) were added to the plate at 1:30,000 and incubated f or 1 h at 37ºC. Plates were washed between each step with 0.05% Tween 20 in PBS. Plates were developed using either 3,3 ór ,5,5tetramethylbenzidine (TMB; Thermo Fisher Scientific, Waltham, Massachusetts, USA) and read at 450 nm. When testing binding to linear peptides, cyclic V2, or 1J08 V1/V2 scaffolds, a ratio of the molecular weights of these constructs to the native glycoprotein monomer was calculated to obtain coating with the same number of epitopes/well. Competition assays of anti-V2 mAbs were performed by enzyme-linked immunosorbent assay (ELISA) as described by Mason, et al. 

Peripheral blood mononuclear cells (PBMCs) from Rhesus macaques were isolated by centrifugation of EDTA whole blood on a Ficoll-Paque Plus gradient. CD8 + T cells were depleted using CD8 beads (Miltenyi Bio Beads) and stimulated for three days in PHA followed by in vitro infection with SIVmac251.

Cells were maintained in RPMI containing 15% FBS, 1% Penicillin Streptomycin, 1% glutamine, and 40 IU/mL of IL-2 f or at least 3 days at 37ºC, 5% CO2, and P27 levels were assayed by ELISA to measure productive viral replication. Following at least 3 days of culture, infected or naïve cells from the same animal were centrif uged in PBS and resuspended to 1x10 6 cells/mL, and 1x10 6 cells were pelleted in separate FACS tubes.

To assess competition between anti-V1a and V2 antibodies, the cell pellets were resuspended in 100 µL of a 1:1 PBS serial dilution of ITS41 mAb starting at 2.5µg/mL and incubated for 30 min at RT. Cells were washed in 1 mL PBS and centrifuged at 2,000 RPM for 6 min, resuspended in 50 µL of PBS containing 

The ability of the gp120 mutants (WT, ∆V1, or ∆V1gpg) to be bound by V1 and V2 mAbs was measured by ELISA. ELISA plates were coated overnight with 40 µl, 100 ng of SIVmac251-M766/gp120 mutants (gp120WT, ∆V1, or ∆V1gpg) in PBS, washed once with PBS, and blocked with 100 µl of 1% BT3 (150mM NaCl, 50mM (1:2,000) in PBS containing 0.1% Tween 20 and 0.25% milk. Membranes were washed in PBS 0.1% Tween and exposed to a horseradish peroxidase-conjugated goat secondary anti-monkey antibody

(1:10,000; Abcam #ab112767). Immunoreactivity proteins were visualized by chemiluminescence using a ChemiDoc™ Imaging System (Biorad). Densitometric analysis was performed using Image Lab Software.

The immune precipitation of the HIV gp120 proteins (AE.A244 D11gp120 WT or ΔV1) were performed with the same methodology described above. The antibodies used for immunoprecipitation were PG9, an anti-V2 antibody that recognizes V2 in β-Barrel conformation ( 

Gp120 

SIV pseudoviruses were produced as previously described 

Rectal mucosal env-SIV IgG was measured from rectal mucosa swabs collected at 2 weeks before vaccination and at week 14. Swabs were collected from animals in the WT, ∆V1, or ∆V1gpg groups by custom SIV binding antibody multiplex assays (SIV-BAMA) as previously described (Tomaras et al., 2013) . Samples were processed, examined for blood contamination, and measured for semiquantitative evaluation of hemoglobin. The total IgG concentration was measured by a custom macaque total IgG 

We used a static adhesion assay to characterize the interaction between gp120 and α4β7 based on the method developed by Dr. Peachman and colleagues (Peachman et al., 2015) in which RPMI8866 cells, which express α4β7 on the cell surface, were allowed to adhere to the recombinant Env proteins (partly deglycosylated), V1/V2 scaffolds, or synthetic cyclic V2 peptides. The α4β7-expressing RPMI8866 cell line was derived from a human B cell lymphoma, and expresses α4β7, but no detectable CD4. Cells were grown in media containing retinoic acid, which increased the levels of both expression and clustering of α4β7. In some assays, we included anti-integrin (Vedolizumab) and anti-gp120 mAbs or plasma as adhesion inhibitors. For plasma samples considered the cut-off of 15% of binding inhibition, combined with the condition that the percentage of inhibition from induced by plasma from week 17 should be at least 2x higher than the baseline. This cell-based assay measured adhesion between two multivalent surf aces.

To characterize the interaction between gp120 and α4β7, we developed a novel surface-plasmon resonance (SPR) -based assay that utilized dextran surfaces coated with recombinant Env proteins, V1/V2 scaffolds, or synthetic cyclic V2 peptides (Lertjuthaporn et al., 2018) . The analyte that reacted with these surf aces was a recombinant soluble α4β7 heterodimer in which the carboxy-terminal transmembrane and cytoplasmic tail domains of both chains were removed and replaced by short peptides that function as an "α4 chain acid-β7 chain base coiled-coil clasp" (Nishiuchi et al., 2006) . This acid-base clasp was joined by a disulfide bond that served to stabilize the heterodimer. In one iteration of this assay, we employed short, linear peptides derived from V2 as competitive inhibitors.

Antibody-dependent neutrophil phagocytosis (ADNP) ∆V1gp120 was biotinylated following manufacturer's instructions (Thermo Fisher Scientific) and incubated with yellow-green streptavidin-fluorescent beads (Molecular Probes) for 2 h at 37ºC. Here, 10 μl of a 100f old dilution of beads-protein were incubated for 2 h at 37ºC with 100 μl diluted plasma samples before the addition of effector cells (50,000 cells/well). Fresh peripheral blood leukocytes from one healthy donor were used as effector cells after red blood cell lysis with ACK lysing buffer (Thermo Fisher Scientific).

Af ter 1 h incubation at 37ºC, the cells were washed, surface stained, fixed with 4% formaldehyde solution (Tousimis, Rockville, Maryland), and their fluorescence was evaluated on an LSRII (BD Biosciences).

Anti-human CD3 AF700 (clone UCHT1) and anti-human CD14 APC-Cy7 (clone MϕP9) antibodies ADCC CEM-based assay.

We tested the percentage of CEM that reacted with the anti-V2 NCI05 and NCI09 mAbs coated with the gp120 immunogens. EGFP-CEM-NKr-CCR5-SNAP cells were incubated with 50 μg of gp120 protein WT, ΔV1, or ΔV1gpg for 2 h at 37ºC. After wash, coated cells were incubated with 5 μg/ml of NCI05 or NCI09

antibody at RT f or 30 mins. The cells were washed and incubated with secondary IgG anti-monkey antibody conjugated with PE. Uncoated target cells in the presence of NCI05 or NCI09 and secondary antibody were used as negative control. Cells were acquired on a SORP LSR II (BD Biosciences) and analyzed using FlowJo Software (FlowJo, Ashland, OR).

ADCC activity was assessed as previously described using EGFP-CEM-NKr-CCR5-SNAP cells that constitutively express GFP as targets (Orlandi et al., 2016) . Briefly, one million target cells were incubated with 50 μg of gp120 protein wild type, ΔV1, or ΔV1-gpg for 2 h at 37ºC. After this coating, the target cells were washed and labeled with SNAP-Surface® Alexa Fluor® 647 (New England Biolabs, Connecticut, USA S9136S) per manufacturer recommendations for 30 min at RT. Plasma samples, heat inactivated at 56ºC f or 30 min, were serially diluted (7 ten-fold dilutions starting at 1:10) and 100 μl were added to wells of a 96-well V-bottom plate (Millipore Sigma). 5000 target cells (50 μl) and 250,000 human PBMCs (50 μl)

were added as effectors to each well to give an effector/target (E/T) ratio of 50:1. The plate was incubated at 37ºC f or 2 h f ollowed by two PBS washes. The cells were resuspended in 200 μl of a 2% PBSparaf ormaldehyde solution and acquired on an LSRII equipped with a high throughput system (BD Biosciences, San Jose, California, USA). Specific killing was measured by loss of GFP from the SNAP-Alexa647 + target cells. Target and effector cells cultured in the presence of R10 medium were used as background. Anti-SIVmac gp120 monoclonal antibody KK17 (NIH AIDS reagent program) was used as a positive control. Normalized percent killing was calculated as: (killing in the presence of plasmabackground)/ (killing in the presence of KK17-background) X100. The ADCC endpoint titer is defined as the reciprocal dilution at which the percent ADCC killing was greater than the mean percent killing of the background wells containing medium only with target and effector cells, plus three standard deviations.

F(ab')2 f ragments were prepared from NCI04, NCI05 or NCI09 mAb using Pierce f(ab')2 Micro Preparation Kit ( Cat#44688, Thermo scientific) following the manufacturer's instructions. A SDS-page gel with the recovered F(ab')2 was run and Silver stained (Cat# LC6070, Silver Quest staining Kit, Invitrogen)

according to the manufacturer's instructions, to assure the purity of the F(ab')2 fragments. Target cells, coated with gp120 as indicated and labeled with SNAP-Surface® Alexa Fluor® 647, were incubated for 1 h at 37ºC with 6 ten-f old serial dilutions, beginning at 1 μg of purified F(ab')2 f ragments from NCI04, NCI05, or NCI09 monoclonal antibodies. Cells incubated without F(ab')2 served as control. These target cells were subsequently used in the ADCC assay as described above.

Competitive ADCC assay ITS41, a V1 monoclonal antibody, was used to compete with NCI05 or NCI09 -mediated ADCC activity.

NCI04 monoclonal antibody served as an additional control. Six serial ten-fold dilutions of ITS41 and NCI04 were perf ormed in a 96 well V-bottom plate beginning at 50 µg. In addition, 1 µg of the NCI05 or NCI09 antibody was added to each well. Target cells coated with the wild type gp120 protein and labeled with SNAP-Alexa647 together with effector cells were added as described above for the ADCC assay.

ADCC activity in both the presence and absence of competing antibodies was then determined. The experiment was repeated 3 times.

The levels of Neutralizing antibodies were measured in the plasma of animals from the three vaccinated group (WT, ∆V1, and ∆V1gpg) at baseline and week 17 (5 weeks after the last immunization) as a reduction in luciferase reporter gene expression after a single round of infection in TZM-b1 cells as 

SIVmac251 RNA in plasma was quantified by nucleic acid sequence-based amplification, as previously described (Romano et al., 1997) . SIV DNA was quantified in mucosal tissues 3 weeks after infection by a real-time qPCR assay with sensitivity up to ten copies × 10 6 cells, as previously described (Lee et al., 2010) .

The variable region of the NCI09 heavy chain was synthesized and cloned into a pVRC8400 vector containing an HRV3C cleavage site in the hinge region as previously described (McLellan et al., 2011) .

Heavy and light chain plasmids were co-expressed in 1 liter of Expi293F cells. IgG was purified from the supernatant through binding to a protein A Plus Agarose (Pierce) column and eluted with IgG Binding

Buf fer (Thermo Fisher Scientific). Antibodies were buffer-exchanged to PBS, then 10 mg of IgG were cleaved with HRV3C protease. The digested IgG was then passed over a 2 ml protein A Plus column to remove the Fc fragment. The Fab was further purified over a Superdex 200 gel filtration column in buffer containing 5 mM HEPES 7.5, 50 mM NaCl, and 0.02% NaN3. To form NCI09-V2 peptide complexes, 5 mg of purified fab at a concentration of 2 mg/ml were incubated at RT f or 30 min with a f ive-fold molar excess of SIV V2 peptide, synthesized by GenScript, and the complex was then concentrated down to 10 mg/ml using 10,000 MWCO Ultra centrifugal filter units (EMD Millipore). Antibody-peptide complexes were then screened against 576 crystallization conditions using a Mosquito crystallization robot mixing ○ There are 5 unique types of molecules in this entry. The entry contains 7693 atoms, of which 3501 are hydrogens and 0 are deuteriums.

In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.

• Molecule 1 is a protein called NCI09 light chain. 3 Residue-property plots i ○ These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry. The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometry and electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor fit to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.

• • Molecule 4: beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alp ha-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose Xtriage's analysis on translational NCS is as follows: The largest off-origin peak in the Patterson function is 5.02% of the height of the origin peak. No significant pseudotranslation is detected.

5 Model quality i ○ 5.1 Standard geometry i ○ Bond lengths and bond angles in the following residue types are not validated in this section: BMA, NAG, FUC

The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 5 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles). There are no bond length outliers.

There are no bond angle outliers.

There are no chirality outliers.

There are no planarity outliers.

In the following table, the Non-H and H(model) columns list the number of non-hydrogen atoms and hydrogen atoms in the chain respectively. The H(added) column lists the number of hydrogen atoms added and optimized by MolProbity. The Clashes column lists the number of clashes within the asymmetric unit, whereas Symm-Clashes lists symmetry-related clashes. The all-atom clashscore is defined as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 2.

All (15) close contacts within the same asymmetric unit are listed below, sorted by their clash magnitude. 

In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.

The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues. There are no Ramachandran outliers to report.

In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.

The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues. There are no ring outliers.

No monomer is involved in short contacts.

There are no ligands in this entry.

There are no such residues in this entry.

There are no chain breaks in this entry. 6 Fit of model and data i ○ 6.1 Protein, DNA and RNA chains i ○ In the following table, the column labelled '#RSRZ> 2' contains the number (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution. The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue. The column labelled 'Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9. 

There are no such residues in this entry.

Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins

Crystals were flash-frozen in liquid nitrogen supplemented with 20% ethylene glycol as a cryoprotectant. Data were collected at 1.00 Å using the SER-CAT beamline ID-22 of the Advanced Photon Source, Argonne National Laboratory. Diffraction data were processed with an HKL-2000 (HKL Research). A molecular replacement solution obtained from Phenix (www.phenix-online.org) contained one Fab molecule per asymmetric unit in space group P212121. Model building was carried out using COOT software

Optimal characteristics were considered as f olding into an α-helix, and helical stability was assessed by the energy spectrum of the folding. The optimal fragment from SIVmac251 was 14 amino acids in length with sequence DKTKEYNETWYSTD, and its equivalent fragment from SIVSME543 was DKKIEYNETWYSRD. These peptide sequences were designed into probes suitable for ELISA by adding an N-terminal biotin and tri-glycine linker (biotin-GGG-V2c sequence) and synthesized commercially

A244 D11 gp120 ΔV1 protein expression

The sequence of AE.A244 D11 gp120 recombinant protein was obtained from the CRFO1_AE Env gp120

The f irst 11 amino acids at the Nterminus of the mature Env protein have been deleted, as described by

For the purpose of this study, we designed an HIV gp120 mutant protein with deletion of the V1 region (TKANLTNVNNRTNVSNIIGNITD) identif ied here as AE.A244 D11gp120 ΔV1a. The protein/DNA sequence is represented in Table S9. The A244 WT and A244 ΔV1a mutant genes were cloned into pSWTIPK3 (Advanced BioScience Laboratories, Inc.) downstream of the CMV promoter and Kozak sequence

Proteins were purified from the conditioned cell culture supernatant using a lectin-affinity chromatography (Galanthus nivalis lectin agarose; Vector Labs, Inc.) capture step

Statistical analysis Statistical analysis was performed using the Wilcoxon signed-rank test or Mann-Whitney test to compare continuous factors between two paired or unpaired groups, respectively. Comparison between multiple groups was done with the non-parametrical Kruskal-Wallis test with Dunn's multiple comparison test or the 2-way ANOVA test with Tukey's or Dunn's multiple comparison tests. Comparisons of differences between groups in the number of challenges before viral acquisition were assessed using the log-rank (Mantel-Cox) test of the discrete-time proportional hazards model. The average per-risk challenge of viral acquisition was estimated as the total number of observed infections divided by the number of administered challenges. Correlation analyses were performed using the non-parametric Spearman rank correlation method with exact permutation two-tailed P values calculated

This is a Full wwPDB X-ray Structure Validation Report for a publicly released PDB entry. We welcome your comments at validation@mail.wwpdb.org A user guide is

The following versions of software and data (see references i ○) were used

Validation Pipeline

1 Overall quality at a glance i ○The following experimental techniques were used to determine the structure:

The reported resolution of this entry is 1.40 Å.Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based. The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density. The red, orange, yellow and green segments of the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor fit to the electron density. The numeric value is given above the bar.

Quality of chain