Rapid Immunogen Selection (R.I.S.) method

Patent applications WO 2012113921 and 2014027082. i) In-vitro random mutagenesis and subcloning. Random mutations in the HIV env gene were introduced using a Genemorph II Random Mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer’s recommendations and using the AC10_29 sequence as the starting template. For mutagenesis, primers 179 and 180 were used (S1 Table). For the generation of a library of chimeric virions, randomly mutated env genes were inserted into pNL4-3 (obtained from the National Institutes of Health AIDS Research and Reference Reagent Program; NIH ARRRP) using convenient restriction sites (XbaI and NotI) to generate replication competent virions. Randomly mutated env genes were also introduced into pcDNA 3.1+ (Invitrogen; Carlsbad, CA, U.S.A.) using XbaI and NotI restriction sites. pcDNA cloning was chosen for library characterization due to its higher cloning efficiency that has, therefore, a minor cloning associated bottleneck. Five independent ligations and transformations were performed simultaneously to avoid the loss of variability and mixed together in the up scaling of amplification. Env-positive clones were sequenced with primers 183, 185, 186, 190, 192 and 193 (S1 Table). ii) Virion library generation. Virion library was produced by transfecting the DNA constructions in 293T cells using the calcium phosphate method according to the manufacturer´s recommended protocol (ProFection mammalian transfection system; Promega, Madison, WI). iii) Virion capture assay. 96-well plates were coated overnight at 4°C with 0.5μg of goat anti-human polyclonal anti-Fc antibodies in PBS. Next, wells were blocked with 3% bovine serum albumin (BSA) in PBS for 1h at 37°C. Viruses generated from the library were used for selection (20ng of p24) with 4E10 antibody (5μg/well) for 1h at 37°C with agitation (450 r.p.m.) in a round bottom 96-well plate, allowing binding to occur. Virus-Ab complexes and a control virus suspension without antibody were then transferred to blocked microtiter wells previously coated with anti-Fc antibodies. After a 2h incubation at 37°C, wells were washed six times with PBS, and RNA was extracted with High Pure Viral RNA kit by adding the binding buffer directly to the well (Roche, Basel, Switzerland). iv) Nested RT-PCR HIV-1 env RNA amplification. Isolated HIV-1 env RNA was then transferred to an Eppendorf tube and, next, it was amplified by reverse transcriptase polymerase chain reaction (RT-PCR). An RNA template was used for the RT-PCR reaction. RNA was reverse transcribed with primer 102 [30]) at 50°C for 20 min with GeneAmp Gold RNA PCR reagent kit (Applied Biosystems, Waltham, MA, U.S.A.). Env was amplified with the primers 101[30] and 104 [30] from cDNA followed by a nested PCR with primers 179 and 180 (S1 Table) and Expand High Fidelity PCR system (Roche, Basilea, Switzeland). Amplified envs were subcloned and virus stocks were generated as described above.

Deep-sequencing

DNA amplification was carried out by a PCR with primers 454_1 and 454_2 for amplicon 1; 454_3 and 454_4 for amplicon 2 and 454_5 and 454_6 for amplicon 3 (S1 Table and S1 Fig). Primers were further ligated with multiple identifier adaptors (MID) specific to the Titanium sequencing chemistry (454/GS/Junior, Roche Diagnostics, Branford, CT). The amplicon library was generated in triplicate followed by pooling and purification of triplicate PCR products using magnetic beads (Agencourt AMPure Kit, Beckman Coulter Inc., Benried, DE) to eliminate primer-dimers. The number of molecules was quantified by fluorometry (Quant-iT PicoGreen dsDNA assay kit, Invitrogen Corp., Carslbad, CA, USA). Emulsion PCR (emPCR) was carried out according to the 454 Titanium emPCR protocol (Roche Diagnostics, Branford, CT). The sequencing run was performed on a picotiter plate using the 454/GS/Junior.

Virion-antibody affinity assays

96-well plates were coated overnight at 4°C with 0.5μg of goat anti-human polyclonal anti-Fc antibodies in PBS. Next, wells were blocked with 3% bovine serum albumin (BSA) in PBS for 1h at 37°C. Serial 2-fold dilutions of recombinant viruses were incubated with the corresponding mAbs (5μg/well) for 1h at 37°C with agitation (450 r.p.m.) allowing binding to occur. The virus-Ab complexes and the control virus suspension with no mAb were then transferred to the blocked microtiter wells previously coated with the immobilized anti-Fc antibodies. Control virus suspension with no mAb showed the background level of non-specific binding. After a 2h incubation at 37°C, wells were washed six times with PBS, and virus equivalents were quantified by p24 ELISA. Statistical analysis was conducted by R using One-way NOVA followed by Newman Keuls post hoc test (***p<0.001 and *p<0.05). Data are represented as mean ± SDEV.

Specific envelope amino acid analysis

Gp160 sequences were downloaded from CATNAP database in FASTA format (http://hiv.lanl.gov/catnap) together with IC₅₀ values for each antibody-virus pair [31]. The analysis was performed with 675 antibody-virus pairs for 4E10, 676 pairs for 2F5, 144 for 447-52D, 639 for VRC01, 669 for 2G12, 423 for PG16, 255 for PGT151 and 715 for b12. HIV-1 isolates with W503 and N643 amino acids could not be included in the analysis because there was no neutralization data for these isolates in the database. IC₅₀ geometric mean of undetected neutralization was set to 100μg/ml, as described at CATNAP database. Statistical significance was evaluated with a Mann-Whitney U test. Simple comparisons were made with the use of a two-sided alpha level of 0.05.

Optimization of Gag sequence as a T-cell immunogen

An artificial full-length Gag sequence, referred to as dGag was used for the formation of VLP structures. The dGag sequence is based on the 2001 HIV-1 clade B Gag consensus sequence available at the Los Alamos National Laboratory HIV database with modifications at 32 amino acid positions. These sequence changes were introduced to complement our in-house developed T cell immunogen sequence [32] at positions that are frequent targets of the virus-specific CD8 T cell responses (HIV database: https://www.hiv.lanl.gov; [33]) and include residues with more than 10% frequency in the clade B sequence alignment. No changes were introduced in the oligomerization region in p24.

Generation of virus-like particles

HIV-1-dGag-VLPs, composed of the HIV-1 dGag sequence, were produced by transient transfection with the FreeStyle 293 Expression System (Invitrogen, Carlsbad, CA, USA) co-transfecting with a pcDNA-dGag plasmid and codon optimized pcDNA3.1-AC10_29 or pcDNA3.1-LR1C1 envelope expression plasmids provided by GeneArt Gene Synthesis (ThermoFisher Scientific, Waltham, MA, USA). For transfection, 1μg DNA/10⁶cells of each HIV-1 dGag or HIV-1 Env-expression plasmids were used. Different ratios of dGag/Env expression plasmids were tested and 3:1 and 10:1 ratios were chosen for AC10_29 and LR1-C1 VLPs respectively in order to obtain similar Env incorporation for each VLP. At 48 hours post-transfection, VLPs were harvested by ultracentrifugation [34]. Briefly, VLP supernatants were first clarified through centrifugation twice at 800g for 5 min and then ultracentrifugated at 50000g for 32 min. Pellets were ultracentrifuged once more at 160000g for 10 min and resuspended in Trehalose 5%/PBS to preserve VLP integrity and stored at -80°C [35].

Immuno-electron cryo-microscopy

VLPs were produced and ultracentrifugated as described above. VLP pellets were fixed with a 4% paraformaldehyde 0.1% glutaraldehyde solution in 0.1M PB for 1h at 4°C. Next, the solution was removed carefully and pellets were stored at 4°C in a 2% paraformaldehyde in 0.1M PHEM at 37°C and cryo-preserved in 2.3M sucrose in 0.1M PHEM at 4°C overnight. Then, they were mounted in pins and frozen in liquid nitrogen. Ultrathin sections were obtained in a FC7 ultramicrotome (Leica Microsystems, Wetzlar, Germany) and were immunolabeled with an anti-gp120 antibody (447-52D; Polymun Scientific, Klosterneuburg, Austria) and a gold-conjugated anti-human antibody (Jackson Immunoresearch, West Frove, PA, USA). Samples were visualized in a Transmission Electron Microscopy (TEM) Tecnai Spirit (FEI) at 120 kV with a LaB6 cathode and equipped with a 1kx1k Megaview CCD Camera.

Western blotting

Env incorporation per virion or per VLP was analysed by western blotting in SDS-PAGE gels. Identical quantities of p24 or p55 were mixed with Laemmli buffer supplemented with 5% β-mercaptoethanol, boiled for 10 minutes and kept on ice. Env expression in 293T cells was also analysed by western blotting after lysing with a Cell lysis buffer (Cell Signaling Tecnology, Danvers, MA, U.S.A) and boiled for 10 minutes. Samples were then electrophoresed at 160V for 90 minutes through a 4 to 12% polyacrylamide-sodium dodecyl sulfate gradient gel (GE Healthcare, Chicago, IL, USA). Following electrophoresis, proteins were transferred in a semi-dry system onto a PVDF membrane for 45 minutes at 0,1mA. Membranes were blocked with 5% skim milk in Tris-buffered saline tween 20 (TBST) for 1h at room temperature. Membranes were then incubated with antibodies recognizing the gp120 (447-52D provided by the NIH AIDS Research and Reference Reagent Program), Gag (p24 and p55) [p24 Antibody (24–4); Santa Cruz Biotechnology, Santa Cruz, CA, USA] and β-actin [β-Actin (13E5) Rabbit mAb; Cell Signaling Technology, Danvers, MA, U.S.A.). A goat anti-human-HRP (SC-2907; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to detect gp120. Finally, Env and Gag proteins were detected with Clarity Western ECL (Bio-rad, Hercules, CA, USA) using the ImageQuant LAS 500 imager (GE Healthcare, Chicago, IL, USA). Quantitative western blot was performed as described previously [36] with a standard curve of p24 recombinant protein (HIV-1/NL43 p24 capsid p1M) to quantify p55 in VLP samples. The presence of trimers was analysed by western blotting in BN-PAGE gels as described previously [37]. VLPs were solubilized at room temperature for 10 min in 0.12% Triton X-100 in 1mM EDTA-1.5 aminocaprotic acid with 1μl of protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) at 1:1 ratio. The sample buffer used was 100mM MOPS, 100mM Tris-HCl pH: 7.7, 40% glycerol and 0.1% coomassie Blue G250 added at 1:1 ratio. Samples were loaded onto 4–12% Bis-Tris NuPAGE (Invitrogen, Carlsbad, CA, USA) separated for 3h at 100V at 4°C using High Molecular Weight calibration kit for native electrophoresis (Amersham, Buckinghamshire, UK) as a size standard. Proteins were blotted in a wet tank onto a PDF membrane at 4°C and 30V overnight. Then, membranes were blocked with 4% nonfat milk/PBS buffer for 1 hour at room temperature and stained with an anti-gp120 and anti-gp41 antibody cocktail (MAbs 2G12, b12, 39F, 2F5, 4E10 and 447-52D at 1μg/ml each one) overnight at 4°C. Finally, membranes were washed 3 times with PBS, probed by an anti-human-HRP (Jackson ImmunoResearch, West Grove, CA, USA) and proteins were detected by Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Immunization of BALB/c mice

The viral stocks used for immunization were obtained by transient transfection in 293T cells as described above. Viral stocks were inactivated by incubation with Aldritiol-2 1mM at 37°C for 2h under continuous agitation. Female BALB/c mice aged 6 to 8 weeks and specific pathogen free (SPF) were utilized. Mice were fed ad libitum with a standard diet and kept under a light-dark cycle of 12h. Each group was composed of 5 animals. Both groups were inoculated with 100μl of AT-2 inactivated virus (2.5μg/ml p24 quantified as explained above) and 100μl of complete Freund adjuvant subcutaneously divided among all 4 legs (t = 0). Two weeks later (t = 14 days), mice were inoculated with 100μl of AT-2 inactivated virus (2.5μg/ml p24) and 100μl of incomplete Freund adjuvant subcutaneously into each leg. After two weeks (t = 28 days), between 400μl and 1ml of blood were extracted by cardiac puncture under isoflurane anesthesia from both groups. Mice euthanasia was performed by overdose of isoflurane. One subject from the first group perished before conclusion of the four weeks for reasons not related to the experimental protocol (S4 Fig). These experiments were performed in a BSL3 Biocontainment Facility at Centre de Recerca en Sanitat Animal (CReSA) with the support by CReSA staff (Barcelona, Spain).

Rabbit immunizations

5 groups of rabbits were immunized (4 per group): HIV-1-Gag-VLPs as a control group (G1); HIV-1-Gag-VLPs with AC10_29 Env (G2/G4) and LR1-C1 Env (G3/G5). In G1-G3 groups Adjuplex adjuvant (Sigma-Aldrich, St. Louis, MO, USA) was used while no adjuvant was added in groups G4 and G5. The vaccination protocol used was a DNA prime followed by one DNA boost (500μg of DNA/boost/animal) and 3 VLP boosts (20μg of p24 of VLP/boost; S5 Fig). 15 weeks after immunization, the presence of anti-HIV-1 antibodies was analyzed using New Lav Blot I kit (Bio-Rad, Hercules, CA, USA) substituting the horseradich peroxidase-conjugated for a alkaline phosphatase-goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) diluted 1/1,000 in 5% milk in PBS. All rabbits were maintained in 785 x 840 x 1840 h (mm) cages with access to water and food ad libitum and under constant humidity and temperature with a light/dark cycle of 12 hours. All procedures were conducted in accordance with the guide-lines established by the University of Barcelona´s Bioethics Committee, as stated in Law 214/97 (July 30^th) drawn up by the Generalitat de Catalunya.

Enzyme-linked immunoabsorbent assays (ELISA)

Wells of 96-well high-binding plates (Inmulon, ThermoFisher Scientific, Waltham, MA, USA) were coated with 100μl of AC10_29 (HIV-1, clade B; gp120; Abnova, Tipei, Taiwan) at 200ng/well with phosphate-buffered saline (PBS) and incubated at 4°C overnight. Next, wells were blocked with 150μl of 5% nonfat powdered milk in PBS at 37°c for 1h. 100μl of the corresponding sera dilutions in 5% PBS-milk were added to each well and incubated at 37°C for 1h. After washing three times with PBS plus 0.05% Tween 20, 100μl of horseradich peroxidase-conjugated goat anti-rabbit immunoglobulin G antibody (sc-2004, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1/2,000 in 5% milk in PBS was added to each well, and the plates were incubated at 37°C for 1h. Plates were then washed 6 times with PBS-Tween, and 100μl of tetramethylbenzidine reagent (Calbiochem, San Diego, CA, USA) was added to each well. 10 minutes later, 50μl of hydrochloric acid was added to each well, and optical density at 450nm was measured using a spectrophotometer (Sunrise, TECAN, Zürich, Switzerland). For Env peptide ELISA, 15-mer peptides corresponding to the V3 loop and the gp41 ectodomain of AC10_29 Env sequence (283 to 341 amino acids and 629 to 686 respectively, numbered according to HXB2) were synthesized by Bionova (V3 peptides; Bionova, Halifax, Canada) and by the Unit of Synthesis and Biomedical Applications of Peptides, IQAC-CSIC, Barcelona (gp41 peptides). The assay was performed as described previously [38].

Neutralization assays

400 μl of sera from immunized mice and rabbits were pre-adsorbed with 1x10⁶ cells that were used for virus or VLP production (293T and 293F respectively) for 1h at room temperature with rotation. Next, IgGs purified from pre-adsorbed immunized animal sera were purified with prot A columns (GE Healthcare, Chicago, Il) and tested against a panel of 6 recombinant viruses from five different subtypes as described previously [39].

Generation and characterization of a randomly mutated envelope library (L)

The objective of this study was the selection of Env variants with increased exposure of epitopes recognized by bNAb 4E10. This is an early antibody candidate that was available at the time of initiating selection experiments and humoral immunity to its targeted region had been associated with broad antiviral activity [40]. AC10_29 envelope from a clade B, tier 2 primary isolate was selected as the template for generating the randomly mutated library. AC10_29 was chosen for being a tier 2 primary isolate from a recently infected patient [41]. Tier 2 neutralization phenotype has been associated to closed Env trimer configuration that is believed to be more relevant for vaccination and is typical of most circulating strains [42]. AC10_29 overall neutralization profile against a group of bNAbs, including 4E10, is shown in S2 Table and our data are consistent with the data available at the CATNAP database (http://hiv.lanl.gov/catnap) [31]. Random mutagenesis was performed in the env gene by a PCR based method with conditions chosen in order to generate a mutation frequency of approximately 10⁻³ substitutions per nucleotide (Fig 1A). To monitor the mutations accumulated during random mutagenesis, the library (L) was cloned into pcDNA 3.1+ and the full-length env genomic nucleotide sequence from 15 clones was determined (Table 1). The frequencies of each mutation ranged between 22.3% for G→A and 1.5% for A→C. Non-synonymous mutations represented 47% of the total changes introduced. An insertion of one nucleotide and three single nucleotide deletions were also observed among the 15 clones (Table 1). In summary, 215 mutations were found (median; 14.33 and ranging from 3 to 21 mutations per env gene). Overall, mutation frequency in the library (L) was 5.5x10^-3 substitutions per nucleotide calculated by comparison with the consensus sequence that matches AC10_29 and changes were randomly distributed throughout the env gene.

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Fig 1. Rapid Immunogen Selection (R.I.S.) method.

(A) Random mutations were introduced into HIV-1 env AC10_29 sequence by mutagenic PCR. (B) A library of chimeric HIV virions was generated by cloning randomly mutated envelopes into pNL4-3 using XbaI and NotI restriction sites and transient transfection into 293T cells. (C) Virion library was used in an enhanced virion capture assay modified from Leaman et al as indicated in the Materials and Methods section (41). (D) RNA from captured virions was extracted, retrotranscribed into DNA and amplified by nested RT-PCR. (E and B) A new library of chimeric virions was generated by transient transfection of the env amplified product out of captured virions and cloned into NL4-3 proviral DNA.

https://doi.org/10.1371/journal.pone.0208345.g001

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Table 1. Envelope gene mutations in virion libraries determined by standard and deep-sequencing before and after selection.

https://doi.org/10.1371/journal.pone.0208345.t001

Deep-sequencing analysis was used to characterize the library (L) in more detail, as previously described [43]. More than 9,000 454-sequence reads were obtained for each of the 3 amplicons generated from the parental clone and the library (S1 Fig). Considering the 454 sequencing error profile, diversity analysis was performed by counting non-reference nucleobases per nucleotide position on a reference-guided pair wise alignment. Positional diversity, defined as the frequency of all non-reference nucleobases at each position, for the parental clone was invariably below 0.3% except in two neighboring positions located in a homopolymeric region, which showed values around 0.6% (Fig 2A). Positional diversity analysis of the library (L) confirmed a random distribution of mutations in the dataset with no positional diversity higher than 3% (Fig 2B). The frequencies of each type of mutations observed by deep-sequencing were very similar to the frequencies obtained by standard sequencing (Table 1A and 1B). The only significant differences between standard and deep-sequencing analysis were the frequencies of T →G mutation (5.6% vs. 0.0%) and C→T (5.1% vs. 21.1%) (Table 1A and 1B). To determine whether the differences observed in both analyses could be attributed to the different regions sequenced by both methods (the full-length env gene for the standard sequencing and the regions corresponding to the three amplicons for deep-sequencing; S1 Fig), we compared the sequences only in the regions corresponding to the indicated deep-sequencing amplicons and similar results were observed (S3 Table). For this reason we believe that the observed differences could be attributed to cloning efficiency or to the limited number of clones analyzed by standard sequencing. In future studies we will increase the size of the sequenced library to address this issue.

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Fig 2.

Positional diversity detected by deep-sequencing in parental virus AC10_29 (A) and the mutant library before [L; (B)] and after selection by the R.I.S. method [4E10sL; (C)]. (A) Amplicon sequencing of the original primary isolate before random mutagenenesis of the env gene revealed a homogeneous population. (B) The library generated by random mutagenesis had a wide variability of mutations across the amplicons sequenced. (C) Some mutations are fixed after selection with 4E10 by the R.I.S. method. Positional diversity is calculated as the frequency of all nucleotides that differ from the parental strain (AC10_29) at each evaluated position. Insertions and deletions are taken into account. Genomic regions corresponding to each amplicon are indicated.

https://doi.org/10.1371/journal.pone.0208345.g002

Mutation accumulation as result of selection of HIV-1 envelope mutants using 4E10 as bait with the Rapid Immunogen Selection method (R.I.S.)

To determine which mutations accumulated after 4E10 selection out of the randomly mutated env library (L), the 4E10 selected library (4E10sL) was cloned into pcDNA3.1+ and the full-length env genomic nucleotide sequence from 7 clones was determined. Mutation frequencies ranged between 26.6% for A→G and 0.0% for A→C and C→G; Non-synonymous mutations represented 90.4% of the total, in contrast to what was observed in the initial library (47%), which suggests a strong selection force by 4E10 selection. Three single nucleotide deletions were also observed (Table 1). In summary, 79 mutations were found (median: 11.29 and ranging from 8 to 22 mutations per env gene analyzed). Overall, mutation frequency in the library after 4E10 selection (4E10sL) was 4.3 x10^-3 substitutions per nucleotide calculated by comparison with the corresponding parental sequence (AC10_29). Standard sequences have been deposited in GeneBank (accession numbers MK034104 to MK034126)

Next, a deeper analysis of the selected library (4E10sL) was carried out by deep-sequencing. 4E10 antibody-selected env sequences showed a significant increase of specific mutations compared to the initial clone that was used as template (Fig 2C). Deep sequencing experimental design was adapted to preliminary standard sequencing that revealed major differences in the deep-sequenced regions. Deep Sequencing rawdata have been deposited in CBI/SRA repository with accession PRJNA495005. Most of the amino acid replacements observed in the library after selection (4E10sL) with frequencies above 5% are apparently well tolerated by the envelope protein, as indicated by the acceptability value (Table 2). The degree of acceptability of the amino acid substitution follows the criteria described in Feng et al. where interchanges are more likely to happen between amino acids that have similar chemical structures [44]. The acceptability scale ranges from 0 to 6, with the latter value representing replacement by the same amino acid. Only 3 out of the 37 amino acid replacements observed with a frequency above 5% (D138Y, R503W and K574M) have an acceptability value below 3, supporting the notion of poorly accepted mutations. It is important to note that one third of the mutations enriched to above 5% frequency after selection were located in the gp120 V1 and V2 variable loops (Table 2).

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Table 2. Amino acid replacements in AC10_29 parental sequence with frequencies above 5% observed in the envelope library after 4E10 R.I.S. selection (4E10sL).

https://doi.org/10.1371/journal.pone.0208345.t002

To create virions carrying selected env sequences after 4E10 selection, the library 4E10sL was subsequently cloned into the pNL4-3 backbone and sequenced. Sequence analysis of the resulting clones revealed that one of the clones, LR1-C1, incorporated a total of 10 amino acid replacements which were present in more than 10% (none of the amino acid replacements were present in a frequency below 10%) of sequences isolated after 4E10 selection (Table 2; LR1-C1 mutations are indicated). It is noteworthy that most amino acid replacements incorporated by LR1-C1 are rare according to their frequency in the HIV database (Table 2). 5 of these amino replacements (50%) were located in the gp120 V1/V2 loop. Mutation C131Y is especially relevant because this substitution eliminates the native disulfide bond between C131 and C157, thereby disrupting the architecture of the V1/V2 loop. Importantly, among the mutations incorporated in LR1-C1, there is a loss of 4 potential N-linked glycosylation sites: N88D, N137D, N160Y and N187D (Asn-X-Ser/Thr, where X can be any amino acid).

4E10 selected mutations incorporated by LR1-C1 increased virion affinity for 4E10, 2F5 and 447-52D monoclonal antibodies

The affinities of LR1-C1 variant for selected monoclonal neutralizing antibodies were compared to the parental virus (AC10_29) using a virion capture assay. The assay was performed in triplicate for each antibody at each virion concentration (Fig 3). The data showed an increased affinity of LR1-C1 virions for 4E10, 2F5 and 447-52D antibodies compared to the parental virus. A statistically significant increase in affinity was also observed for antibody 2F5 only at the highest input concentration (p<0.001). Noteworthy, a statistically significant affinity increase was also observed for antibody 4E10 at two input concentrations (p<0.001) confirming the efficacy and specificity of the selection process. As confirmation of this result we have observed that LR1-C1 also has increased affinity for antibody 10E8 that is specific for the same epitope (S3 Fig). Additionally, a significant increase was observed for 447-52D at two input concentrations (p<0.05 and <0.001 respectively). The increased affinity of 4E10 selected virions for the 2F5 antibody is consistent with the close proximity in which the two antibody epitopes are located inside the gp41 membrane proximal domain. These affinity increases are also in line with several reports that correlated the loss of glycosylation sites and the disruption of the V1-V2 loop architecture with an increased sensitivity to antibody mediated neutralization [38,45–52]. The increase in the affinity for 447-52D, whose epitope is located in the V3 loop, is also consistent with previous reports that showed the masking of V3 by the V1-V2 loop [53–57]. However, the increased binding by 447-52D suggests that LR1-C1 Env may have changed to an open tier 1-like conformation [58]. The affinities for VRC01 and 2G12, in contrast, decreased compared to AC10_29 and no binding to PG16 and PGT151 was observed for the LR1-C1 variant. The lack of binding to PG16 and PGT151 could be attributed to mutations at positions 160 and 503 respectively that have been previously associated to this effect [59,60]. The decreased exposure of additional epitopes, such as VRC01, 2G12, PG16 and PGT151, that could also be relevant for the induction of broadly neutralizing antibodies could be an undesired side effect associated with the selection process. The low affinity of LR1-C1 to quaternary-structure dependent antibodies, such as PG16 and PGT151, suggests that LR1-C1 envelope may be inefficient forming trimers. No differences in the affinity for 5F3 antibody (non-neutralizing; gp41 C34 helix) were observed.

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Fig 3. Effect of LR1-C1 mutations on affinity for selected bNAbs.

Virus stocks were obtained from transfected 293T cells. Increase in Ab binding was determined by comparison of the virus captured by different monoclonal antibodies and quantified by p24 when similar amounts of virus (AC10_29 in red and filled circles and LR1-C1 in blue and filled squares) were used as input in a Virion Capture Assay (VCA) with the Abs 4E10 (MPER), 2F5 (MPER), 447-52D (V3), VRC01 (CD4bs), 2G12 (N332 V3 glycan patch), b12 (CD4bs), PG16 (quaternary; V1-V1 glycan apex), PGT151 (quaternary; gp120-gp41 integrase) and 5F3 (gp41). Similar amounts of virus suspension with no mAb were used as controls for both viruses (AC10_29 in grey and filled circles and LR1-C1 in grey and filled squares). Statistical analysis was conducted by R using One-way NOVA followed by Newman Keuls post hoc test at each input concentration (***p<0.001 and *p<0.05). Data are represented as mean ± SDEV.

https://doi.org/10.1371/journal.pone.0208345.g003

Amino acids incorporated by LR1-C1 are associated with increased sensitivity to 4E10 and 2F5 antibodies in HIV-1 isolates described in the Los Alamos database

First of all we wanted to determine the association of 4E10 selected amino acids with increased neutralization sensitivities to selected monoclonal antibodies by TZM-bl assays. Unfortunately, this analysis could not be done because LR1-C1 was not infectious in TZM-bl cells (S2 Fig). As an alternative approach, we compared the corresponding IC₅₀s between viruses that had one or more of the amino acid replacements incorporated by LR1-C1 (Table 2) and viruses that did not contain any of those amino acids in HIV-1 isolates from Los Alamos database. Our analyses showed that the IC₅₀ titer of 4E10 and 2F5 antibodies were significantly higher among the virus group with one or more LR1-C1 amino acids compared to the group with no LR1-C1 amino acids (p<0.005 and p<0.0001 respectively; Fig 4). However, IC₅₀ titers of 447-52D, VRC01, 2G12, b12, PG16 and PGT151 did not significantly differ between the virus groups with one or more LR1C1 amino acids or no LR1-C1 amino acids (Fig 4). HIV-1 isolates with Y131, W503 and N643 amino acids were not included in the analysis because there were no isolates with these amino acids or no neutralization data in the database.

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Fig 4. Neutralization sensitivity to bNAbs of viral isolates with 4E10-selected aminoacids.

Graphical plot showing the comparison of neutralization sensitivities to several bNAbs of viruses from neutralization HIV-1 database (http://hiv.lanl.gov/catnap) with any of the aminoacid replacements incorporated by LR1-C1 that are indicated in Table 2 (closed grey squares) and with none of the aminoacids incorporated by LR1-C1 (closed grey circles). HIV-1 isolates with Y131, W503 and N643 aminoacids were not included in the analysis because there was no neutralization data or no isolate with this mutation in the database. Mann-Whitney U test was used for comparisons of continuous variables between groups. Simple comparisons were made with the use of a two-sided alpha level of 0.05.

https://doi.org/10.1371/journal.pone.0208345.g004

Effects of mutations incorporated after 4E10 selection on envelope incorporation in virions

Next, we assessed the extent to which mutations incorporated by LR1-C1 affected incorporation of envelope proteins into virions. To this end, infectious virus was produced in 293T cells, chemically inactivated with AT-2 and pelleted from clarified supernatant. Virion samples were then subjected to analysis by Western blot (Fig 5A). This analysis revealed a significant amount of virions with incomplete processing of Gag precursors evidenced by the presence of p55 and p41 Gag proteins. This effect could be attributed to the AT-2 treatment considering that inhibitors of HIV-1 zinc fingers prevent normal processing of Gag precursors [61]. In addition, we determined the Env to Gag (p55+p41+p24) ratio in virions carring the 4E10-selected envelope, LR1-C1, relative to the AC10_29 by densitometry (Fig 5B). This analysis revealed that LR1-C1 envelope was incorporated into virions at higher levels than AC10_29 (2.7-fold increase; Fig 5). Finally, Env expression in 293T virion producer cells was analyzed and showed a 2-fold increase in Env expression per cell that could be, at least in part, responsible for the greater Env incorporation in LR1-C1 virions (Fig 5C and 5D).

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Fig 5. Effect of different mutations in Env on incorporation into virions.

Viruses were produced by transfection into 293T cells, virions were chemically inactivated with AT-2 and pelleted from the clarified supernatants. (A) SU (gp120) and Gag (p55, p41 and p24) proteins were detected by Western blotting using 447-52D and anti-p24 monoclonal antibodies. (B) Ratios of SU to Gag (p55+p41+p24) were calculated by using phosphorimaging analysis. Virion producer cells were lysed as indicated in the Materials and Methods section. C) SU and β-actin proteins were detected by Western blotting using 447-42D and anti-β-actin monoclonal antibodies. D) Ratios of SU to β-actin were calculated by phosphorimagiing analysis. Data for LR1-C1 are presented relative to the ratio found for AC10 _29 virions.

https://doi.org/10.1371/journal.pone.0208345.g005

4E10-selected envelope variant (LR1-C1) increases the neutralization capacity of the humoral response induced in BALB/c mice

Changes in the humoral immune response following immunization with the AC10_29 wild type or the LR1-C1 variant were evaluated in mice vaccinated with AT-2 inactivated virions. AT-2 inactivation was chosen over heat-inactivation because this procedure better preserves Env three-dimensional structure [62]. In order to deplete the sera of vaccinated animals from the antibodies generated against cellular components present in virions (from 293 producer cells), sera from immunized mice were pre-adsorbed against 293T cells. In order to minimize the background, IgGs were purified from immunized mice and were tested against a VSV pseudotyped HIV-envelope deficient control. Purified IgG fractions were then tested for neutralizing activities against two different HIV-1 strains (NL4-3, tier 1 and AC10_29, tier 2). Assays were run in triplicate at a 0.2mg/ml IgG concentration and neutralization results are shown in Table 3. IgGs purified from mice immunized with LR1-C1 showed a moderately improved neutralization breadth compared to the AC10_29 immunized mice. Three out of the five sera were capable of neutralizing AC10_29 and one of them could also neutralize NL4-3. In contrast, only one mouse of the 5 animals immunized with AC10_29 neutralized AC10_29 virus with no neutralization of NL4-3 at all. IgG purified from immunized mice sera showed no neutralization of the VSV pseudotyped virus used as specificity control. Due to limited sample size, insufficient purified IgGs were obtained from immunized mice and complete neutralization curves could not be performed. Therefore, IC₅₀ values could not be determined and further neutralization testing of against additional HIV isolates and epitope mapping could not be performed.

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Table 3. Neutralization of AC10_29 and LR1-C1 immunized mice.

https://doi.org/10.1371/journal.pone.0208345.t003

4E10-selected envelope variant (LR1-C1) increases immunogenicity of the 4E10-proximal region in rabbits

To further evaluate the humoral immune response induced by the 4E10-selected envelope, rabbits were immunized with HIV-1-Gag virus like particles carrying AC10_29 and LR1-C1 envelope variants. VLPs were generated using the dGag sequence and each envelope sequence as indicated in the Materials and Methods section. Cryo-immuno Transmission Electron Microscopy (TEM) was conducted to analyze VLPs produced from 293F cells that had been transiently transfected with Gag and Env expression vectors as indicated in the Materials and Methods section. Immuno-electron cryo-microscopical analysis revealed that VLPs were spherical with electron density below the VLP membrane. In addition, VLPs resulted in particle sizes around 130nm in diameter and envelope incorporation was verified by 447-52D labelling (Fig 6A). The amount of labeling per VLP was scarce, as is expected of HIV envelope proteins with untruncated gp41 cytoplasmic tails [63]. Transfection conditions were adapted to ensure that both VLPs had similar amounts of envelope incorporated and was verified by western blotting (Fig 6B). The presence of either envelope trimers incorporated into VLPs and was confirmed by BN-page (Fig 6C) showing comparable levels of AC10_29 and LR1-C1 in these VLP preparations.

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Fig 6. Characterization of the VLPs used for rabbit immunization.

VLPs were produced by transfection into 293F cells and VLPs were pelleted from the clarified supernatants. (A) VLPs were analyzed by immunocrioelectromicroscopy labelling with an anti-gp120 antibody, 447-52D. (B) Envelope incorporations in AC10_29 and LR1-C1-VLPs were evaluated by the gp120 and p55 ratios by western blotting in SDS-PAGE gels using 447-52D and p24 (24–4) monoclonal antibodies. (C) Relative Env incorporation into VLPs was calculated by the ratios of gp120 to p55 using phosphorimaging analysis in triplicate. (D) The presence of trimers into AC10_29 and LR1-C1-VLPs and AC10_29 and LR1-C1-virions were analyzed by western blotting in BN-PAGE gels. The different forms of Env were labelled using a anti-gp120 and anti-gp41 cocktail (2G12, b12, 39F, 2F5, 4E10 and 447-52D). As controls, a 96ZM 651_HXB2_V3 SOSIP and a rgp120 were used.

https://doi.org/10.1371/journal.pone.0208345.g006

Rabbits were immunized with VLPs carrying AC10_29 and LR1-C1 envelopes with and without adjuvant (G2: AC10_29 with adjuvant; G3: LR1-C1 with adjuvant; G4: AC10_29 with no adjuvant and G5: LR1-C1 with no adjuvant). Adjuvanted HIV-1-Gag VLPs with no envelope incorporated were used as controls (G1). All rabbits immunized with AC10_29 and LR1-C1 HIV-1-Gag-VLPs developed responses to HIV-1 Gag and Env HIV-1 proteins (Fig 7). Responses to monomeric rgp120 were more potent to both autologous and heterologous rgp120 when Adjuplex was used for immunization (G2/G3; Fig 7B and Fig 7C). In addition, the gp120 responses were slightly higher in the LR1-C1-immunized group compared to the AC10_29 immunized group only with autologous gp120 but these differences were statistically significant only in the groups immunized with no adjuvant. All 8 sera from G2 and G3 rabbits showed antibody responses to the gp120 V3 loop (Fig 8A) and one of the rabbits immunized with LR1-C1 HIV-1-Gag-VLPs (1650) developed an antibody response to a region in close proximity to the 4E10 epitope (18 amino acids upstream; Fig 8B, specifically against C-terminal heptad repeat (CHR) region of gp41 protein). No neutralizing activity against any of the viruses tested was observed in purified IgGs from the immunized rabbit sera.

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Fig 7. Characterization of the humoral responses induced in immunized rabbits.

The humoral immune response of the immunized rabbits was characterized at week 15. (A) The presence of anti-HIV antibodies in rabbit sera were analyzed by western blot. Preimmune sera of rabbit 1640 was also included as an additional negative control. Endpoint ELISA titters to autologous (AC10.29; B) and heterologous (Bal; C) rgp120. Open symbols correspond to animals immunized with no adjuvant. The highest dilution which gave an OD₄₅₀ 2.5 folds higher than pre-immune sera without dilution was designated as the antibody endpoint titter. Results showed data of three independent experiments and results were expressed as the mean ±SD (n = 4). Ns:non significant; *p<0.05 Mann-Whitney U test.

https://doi.org/10.1371/journal.pone.0208345.g007

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Fig 8. Characterization of the antibody responses against the V3-loop and the gp41 ectodomain regions.

(A) Reactivity of sera from immunized rabbits to the V3 loop was analyzed by ELISA of overlapping 15-mer peptides corresponding to the V3 loop of the AC10_29 isolate. Peptide sequences are shown and the 447-52D epitope is indicated. (B) Reactivity of sera from immunized rabbits to the gp41 ectodomain was analyzed by ELISA of overlapping 15-mer peptides corresponding to the gp41 ectodomain of the AC10_29 isolate and one peptide with the LR1C1 sequence. Peptide sequences are shown and the 4E10 and 2F5 epitopes are also indicated.

https://doi.org/10.1371/journal.pone.0208345.g008