Open Access

Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate

  • Maria Luisa Visciano1,
  • Maria Tagliamonte1,
  • Guillaume Stewart-Jones2,
  • Leo Heyndrickx3,
  • Guido Vanham3,
  • Marianne Jansson4,
  • Anders Fomsgaard5,
  • Berit Grevstad5,
  • Meghna Ramaswamy6,
  • Franco M Buonaguro1,
  • Maria Lina Tornesello1,
  • Priscilla Biswas7,
  • Gabriella Scarlatti7,
  • Luigi Buonaguro1Email author and
  • NGIN Consortium
Journal of Translational Medicine201311:165

https://doi.org/10.1186/1479-5876-11-165

Received: 26 March 2013

Accepted: 20 June 2013

Published: 8 July 2013

Abstract

Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutralizing activity against homologous and heterologous viruses. In the present study, we generated a recombinant trimeric soluble gp140, derived from a previously identified Ugandan A-clade HIV field isolate (gp14094UG018). Antibodies elicited in immunized rabbits show a broad binding pattern to HIV envelopes of different clades. An epitope mapping analysis reveals that, on average, the binding is mostly focused on the C1, C2, V3, V5 and C5 regions. Immune sera show neutralization activity to Tier 1 isolates of different clades, demonstrating cross clade neutralizing activity which needs to be further broadened by possible structural modifications of the clade A gp14094UG018. Our results provide a rationale for the design and evaluation of immunogens and the clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV vaccine with broad activity.

Introduction

Major challenges in the development of an HIV vaccine have been the design of immunogens able to induce a strong and sustained immunity with a broad and cross-clade neutralizing activity.

In the course of natural infection, although HIV-1 is highly effective in evading the immune surveillance [13], almost 20% of HIV-infected subjects are able to develop antibodies with a broad degree of neutralization activity, whose role in the disease control, however, is still debated (reviewed in [4]. Such evidences, indeed, suggest that native antigens are able to elicit such bnAbs antibodies. To date, a number of human bnAbs targeting the HIV envelope glycoprotein in its trimeric status have been isolated from HIV-1 infected subjects [514].

Trimeric envelope structures, either soluble or protruding from a membrane-surrounded particle, have been explored as vaccine models for eliciting broadly neutralizing antibodies (bnAbs) [1519].

Indeed, the native and functional HIV-1 envelope glycoprotein (Env) complex is present on the virus surface as a trimer, each of the monomers made of non-covalently loosely associated gp120 surface and gp41 transmembrane glycoproteins [2023]. However, recombinant soluble forms of fully cleaved and functional trimers, have been difficult to obtain for their high instability. On the other hand, the use of gp160 ectodomain (gp140) has led to the production of trimers that can mimic the native Env spike and have shown to be able to elicit neutralizing antibody responses in immunized animals [2427]. These gp140 trimers can be further stabilized by introduction of specific modification in order to strenghten intra- as well as inter-molecular bindings (gp140SOSIP) [28, 29].

We have recently used a similar strategy to present trimeric gp140 Env molecules on HIV Virus-Like Particles produced in both a transient baculovirus expression system [19] and a stably transfected insect cell line [18]. In particular, a gp120 Env molecule derived from a Ugandan HIV-1 isolate of the clade A (94UG018; GenBank accession number AF062521) [30, 31] has previously been shown to induce high Ab titers with cross-clade neutralizing activity in immunized BALB/C mice [3234] and non-human primates [35].

In the present study, the same gp14094UG018 presented on the surface of VLPs has been produced as recombinant soluble trimeric form of Env for evaluation in homologous prime-boost immunization schedules. An immunogenicity study has been performed in rabbits to evaluate the potency and broadness of specific humoral immune response as well as the mapping of the epitope recognition by the Abs elicited by such protein.

Material and methods

Protein immunogen

Recombinant gp140 protein was produced by transient transfection of the pLex-gp14094UG018 plasmid into adherent 293T cells grown in DMEM media supplemented with 10% FCS. Supernatants were collected after 48 hours and fresh media, containing 10% FCS was added to the cells for another 48 hours at which time point the media was collected again. Following centrifugation and filtration of the supernatant through a 0.22 μm filter, the protein was isolated from the pooled supernatants by initial capture using Talon IMAC chromatography, and elution with TBS with 250 mM imidazole followed by GNL Lectin (Vector Labs) capture and eluted with 1M methyl α-D mannopyranoside, 100 mM sodium acetate (pH 4.0) and finally gel filtration with a SD200 column equilibrated with PBS. The protein production and purification was performed using endotoxin-free materials and buffers. The protein was concentrated using a 10 kDa molecular weight cutoff protein concentrator to 1 mg/ml in PBS for immunization.

Immunization protocol

Four female New Zealand white rabbits 6 to 8 weeks old (with a body weight of ~2 kg), were subcutaneously immunized with the trimeric Clade A gp14094UG018 in the presence of the adjuvant CAF01 [36]. Immunizations were performed at week 0, 2, 4 and 8 and each rabbit received 100μg/dose of immunogen. Blood was collected 3 days before the immunization protocol started (pre-immunization) and four (week 12) and six (week 14) weeks after the last immunization. Heat inactivated sera were stored at −80°C until used. All animal handlers were certified in laboratory animal science courses complying with the category B or C requirements of the Federation of European Laboratory Animal Science Association (FELASA). Animal experiments were performed according to the Animal Experimentation Act of Denmark and European Convention ETS 123 (Protection of Vertebrate Animals used for experimental and other scientific purposes).

ELISA assays

Measurement of specific anti-HIV Env IgG antibodies in rabbit serum

The level of anti-envelope specific IgG antibodies in sera of immunized rabbits was determined by ELISA. Five antigens were used: gp14094UG018 (Clade A), gp120 IIIB (Clade B), gp120 W61D (Clade B), gp140 ZM96 (Clade C) or gp140 UG037 (Clade A). Briefly, 100 or 200 μL of each antigen at a final concentration of 1 μg/mL in PBS were directly coated on 96-well MICROTEST assay plates (Becton Dickinson) and plates were incubated overnight at 4°C. Five-fold dilutions (starting from 1:1000) of each rabbit serum were added to the wells and incubated 2 hrs at 37°C. After washing, 100 μL of goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated was added to each well at a concentration of 1 μg/mL. A TMB Ultra 1-step solution (Thermo Scientific) was used to develop the reactions which were stopped after 30 minutes with 100 μl/well of 2N H2SO4. Plates were read at 450 nm. Reactions were considered positive when the optical density (OD) measured by the ELISA was higher than the O.D. + 3SD of the same dilution of pre-immunization sera.

B-cell epitope mapping

B-cell epitope mapping was carried out using 20 mer overlapping synthetic peptides spanning the entire length of IIIB gp120 from C1 to C5 region. For each region 20 mer overlapping synthetic peptides were pooled or singularly coated at a final concentration of 1 μg/ml per each peptide directly on 96-well MICROTEST assay plates (Becton Dickinson) which were incubated overnight at 4°C. Each rabbit serum was added at a dilution of 1:1000 and incubated 2 hrs at 37°C against pools or single peptides. Each tested pool covered a region of gp120. Goat anti-rabbit IgG HRP-conjugated was added to each well at a concentration of 1 μg/mL and the same procedure as indicated above was followed.

Neutralization assay

IgG were purified from heat inactivated (1 hr 56°C) serum using Protein G HP SpinTrap columns (GE Healthcare) according to the manufacturer’s instructions. Eluted IgG fractions were quantified spectrophotometrically (Nanodrop). Neutralizing activity of IgG from immune rabbit sera was evaluated against primary isolates (Bx08, SF162, QH0692, MW965, 92BR025, DJ263.8, 92RW009) through the TZM-bl assay [37]. Briefly, 200 TCID50 of pseudovirus was incubated with four 2-fold dilutions of IgG from 250 μg/ml to 31.25 μg/ml for 1 h at 37°C in a total volume of 100 μl growth medium in 96-well flat-bottom culture plates. Freshly trypsinized cells (1 × 104) were then added in 10% DMEM growth medium containing DEAE-dextran (Sigma) at a final concentration of 15 μg/ml. After 48 h incubation, 100 μl of growth medium was removed from each well and 100 μl of SteadyLite reagent (Perkin Elmer) was added. Luminescence was measured using a Berthold TriStar LB941 luminometer (PerkinElmer). The background controls contained cells only, while the virus controls contained cells plus virus. The percent neutralization for immune rabbit IgG was calculated comparing it to the virus controls. The 50% inhibitory dose (IC50) was calculated as the IgG concentration that induced a 50% reduction in RLU compared to the virus control wells, after subtraction of cell control RLU. Pre-bleed sera were used as negative controls. TriMab, a mix of 3 mAbs (b12, 2G12 and 2F5), (obtained from Centre for AIDS Reagents, NIBSC, UK) was used in every neutralization experiment as a strongly neutralizing control IgG.

Results

Production of soluble trimeric gp14094UG018

The recombinant gp140 protein was expressed at approximately 0.5 mg/liter of DMEM media from transiently transfected 293T cells, and protein expression was expanded to approximately 10 litres. Following a size-exclusion chromatography, fractions corresponding to the gp140 trimer were collected and pooled. Protein purity was analysed by SDS PAGE and shown to be >95% following the final size-exclusion step (Figure 1).
Figure 1

Analysis of the gp140 94UG018 protein. (A) The trimeric form of recombinant gp140 protein was verified and purified by a size-exclusion chromatography. (B) Fractions corresponding to the gp140 trimer were pooled and analyzed by Coomassie stained denaturing SDS PAGE to confirm the purity of the product.

Induction of anti-gp140 specific antibodies

Induction of a humoral immune response by soluble trimeric Clade A gp14094UG018 was evaluated in sera by ELISA performed on microwell plates coated with the monomeric form of the protein used for immunization protocol. Pre-immunization, week 12 and week 14 sera were tested and specific anti-gp140 antibodies were identified in sera from immunized rabbits, with a response stronger at week 12 than at week 14 (Figure 2). Indeed, all four sera from immunized animals at week 12 show a binding to the gp14094UG018, at the 1:625,000 dilution, statistically significant higher than pre-immunized sera (week 0) (p < 0.01). On the contrary, at the same serum dilution, only one (Rb 49381) of the immunized sera at week 14 show a binding statistically significant higher than pre-immunized sera (week 0) (p < 0.05). Moreover, a 50% of maximal binding activity was obtained, on average, with a 1:2.25 × 105 dilution of week 12 serum and a 1:6.5 × 104 dilution of the week 14 serum (p < 0.01), indicating the elicitation of a strong anti-gp140 antibody response four to six weeks after last immunization. Pre-immunization sera showed no positive reactions at any of the dilutions tested.
Figure 2

Evaluation of IgG titers elicited in sera of immunized rabbits against the homologous trimeric gp140 94UG018 . Five-fold dilutions of heat inactivated sera from pre-immunized (week 0) and immunized rabbits (week 12 and 14) were evaluated in ELISA for their reactivity with homologous gp140. Week 12 and 14 correspond to 4 and 6 weeks after the last antigen administration. The 50% binding is indicated for the week 12 and 14 sera.

Determination of broadness of humoral immune response to Env

In order to evaluate the broadness of the binding characterizing the sera from animals immunized with the trimeric gp14094UG018, pre-immune (week 0) and immune sera (week 12) were tested in ELISA against the recombinant HIV gp120/gp140 proteins of different clades, i.e. IIIB (Clade B), W61D (Clade B), ZM96 (Clade C) and UG037 (Clade A). These proteins show, along the entire gp120 sequence, a pattern of similarity with our Ugandan glycoprotein in particular in the constant regions (i.e. C1, C2 and C5) and significantly lower in the variable regions (Figure 3). This is observed also for the UG037, which is of the same clade (Figure 3).
Figure 3

Analysis of sequence homology. The gp120 amino acid sequence of the indicated HIV isolates were aligned with the 94UG018 clade A Ugandan isolate. Sequence homology between each isolate vs the 94UG018 isolate is indicated along the whole gp120 sequence.

Sera from all immunized animals showed a high antibody titer at week 12 against all tested proteins, with a ranking of UG037 = ZM96 > W61D > IIIB (p < 0.01) (Figure 4). In particular, results show that 50% binding activity was obtained, on average, with a 1:2.25 × 105 dilution for the UG037 and ZM96 proteins, exactly the same as for the homologous 94UG018 protein (Figure 2), a 1:1.25 × 105 dilution for the W61D protein and a 1:4.5 × 104 dilution for the IIIB protein (Figure 4). The ELISA results clearly show that immunization with the trimeric gp14094UG018 induces an immune response with a broad binding activity, recognizing HIV gp120 molecules from different clades.
Figure 4

Evaluation of IgG titers elicited in sera of immunized rabbits against heterologous HIV gp120. Five-fold dilutions of heat inactivated rabbits sera, collected at 4 weeks after the last antigen administration (week 12), were evaluated in ELISA for their reactivity with heterologous gp120s. The 50% binding is indicated.

Evaluation of envelope epitopes recognized by the immune sera

In order to identify the main gp120 epitopes recognized by the immune sera elicited by the trimeric gp14094UG018, epitope mapping was carried out using 20 mer overlapping synthetic peptides spanning the entire length of gp120 from the C1 to the C5 region. A tiled array of 47 synthetic peptides (20 aa each) overlapping by 10 residues covering the entire length of gp120 (Table 1) was used for epitope mapping. The heatmap is derived on the O.D.450 values obtained at ELISA reacting sera from each animal to peptides: the higher is the O.D. value (e.g. higher affinity) the darker is the color in the heatmap. Results, showed that the 12 week sera from all immunized animals bound the C1, C2 and C5 constant regions with high affinity and the V2, V3 and V5 variable regions with lower affinity (Figure 5A). The remaining C3 and C4 constant regions as well as the V1 and V4 variable regions were bound with limited affinity or not bound at all. These results strongly correlated with the percentage of divergence between the sequence of the gp14094UG018 and the peptides used as targets in the ELISA (Figure 5B). Nevertheless, it is relevant to note that the immunized animals showed a significant breadth of binding to the target epitopes, suggesting that the same vaccine molecule is able to elicit distinct patterns of antibodies focusing on different epitopes.
Table 1

Inhibitory concentration (IC) 50 for each animal to each viral isolate evaluated in the study

Virus

Week 12

Week 14

 

49378

49381

49384

49391

49378

49381

49384

49391

SF162

62.5

31.25

62.5

31.25

125

125

125

125

BX08

62.5

62.5

125

31.25

250

62.5

>250

>250

MW965

62.5

62.5

62.5

31.25

125

125

125

31.25

DJ263.8

>250

>250

>250

125

250

>250

>250

250

92Br025

>250

>250

>250

>250

>250

>250

>250

>250

92RW009

>250

>250

>250

>250

>250

>250

>250

>250

QH0692

>250

>250

>250

>250

>250

>250

>250

>250

Values are expressed in μg/ml of IgG and represent the reciprocal dilutions.

Figure 5

Heatmap of binding to gp120 peptides’ pool. (A) Intensity of serum binding in ELISA to peptides’ pool covering the constant and variable regions of gp120 is shown as heatmap. Increasing O.D. value, indicating stronger binding, is represented as darker color. (B) Sequence divergence between peptides used as target in ELISA and the 94UG018 clade A Ugandan isolate is indicated along the whole gp120 sequence.

Within the overlapping peptide pools with the highest binding responses, individual overlapping peptides were used as targets for ELISA using sera collected at week 12 (Table 2). The heatmap built on the O.D.450 values shows that, for each rabbit serum, reactivity against individual peptides was not homogeneous, indicating that the reactivity observed against the pools were attributable to specific epitopes (Figure 6). Moreover, most peptides were recognized by each rabbit serum with a broad range of binding efficiency, suggesting that specific regions of the gp14094UG018 have different potency in eliciting an immune response in the evaluated animals (Figure 5 and Table 2) [38]. The regions best bound by antibodies from immunized sera are depicted in an X-ray conformational structure of gp120 (Additional file 1: Figure S1).
Table 2

Overlapping peptides from HIV IIIB used to fine mapping the binding of rabbit sera elicited by the trimeric gp140 Uganda

Gp120 region

Peptide code

Sequence

Gp120 region

Peptide code

Sequence

C1

ARP740-1

ATEKLWVTVYYGVPVWKEATTT

C2

ARP740-24

NGSLAEEEVVIRSVNFTDNA

ARP740-2

VPVWKEATTTLFCASDAKAY

ARP740-25

IRSVNFTDNAKTIIVQLNTS

ARP740-3

LFCASDAKAYDTEVHNVWAT

ARP740-26

VQLNTSVEINCTR

ARP740-4

DTEVHNVWATHACVPTDPN

V3

ARP740-27

VEINCTRPNNNTRKRIRIQ

ARP740-5

HACVPTDPNPQEVVLVNVTE

ARP740-28

NTRKRIRIQRGPGRAFVTIG

ARP740-6

PQEVVLVNVTENFDMWKNDMV

ARP740-29

RGPGRAFVTIGKIGNMRQA

ARP740-7

NFDMWKNDMVEQMHEDIISL

ARP740-30

KIGNMRQAHCNISRAKWNNT

ARP740-8

EQMHEDIISLWDQSLKPCVK

C3

ARP740-31

HCNISRAKWNNTLKQIDSKL

ARP740-9

WDQSLKPCVKLTPLCVSLK

ARP740-32

LKQIDSKLREQFGNNKTIIF

V1

ARP740-10

LTPLCVSLKCTDLKNDTNTN

ARP740-33

REQFGNNKTIIFKQSSGGDPE

ARP740-11

CTDLKNDTNTNSSSGRMIMEK

ARP740-34

KQSSGGDPEIVTHSFNCGGE

ARP740-12

SSSGRMIMEKGEIKNCSFNI

V4

ARP740-35/36

GEFFYCNSTQLFNS

V2

ARP740-13

GEIKNCSFNISTSIRGKVQK

ARP740-37

NSTWFNSTWSTEGSNNTEGS

ARP740-14

STSIRGKVQKEYAFFYKLDI

ARP740-38

TEGSNNTEGSDTTTLPCRI

ARP740-15

EYAFFYKLDIIPIDNDTTSY

C4

ARP740-39

DTTTLPCRIKQIINMWQKVG

ARP740-16

IPIDNDTTSYSLTSCNTSVI

ARP740-40

KQIINMWQKVGKAMYAPPIS

 

ARP740-17

SLTSCNTSVITQACPKVSFE

V5

ARP740-41

KAMYAPPISGQIRCSSNITG

 

ARP740-18

TQACPKVSFEPIPHYCAPA

ARP740-42

GQIRCSSNITGLLLTRDGGNS

 

ARP740-19

PIPHYCAPAGFAILKCNNK

ARP740-43

LLLTRDGGNSNNESEIFRLG

C2

ARP740-20

GFAILKCNNKTFNGTGPCNT

C5

ARP740-44

NNESEIFRLGGGDMRDNWRS

 

ARP740-21

TFNGTGPCNTVSTVQCTHGI

ARP740-45

GGDMRDNWRSELYKYKVVKI

 

ARP740-22

VSTVQCTHGIRPVVSTQLLL

ARP740-46

ELYKYKVVKIEPLGVAPTKA

 

ARP740-23

RPVVSTQLLLNGSLAEEEVV

ARP740-47

EPLGVAPTKAKRRVVQREKR

Bold characters indicate the peptide with the strongest binding by immune sera.

Figure 6

Heatmap of binding to individual gp120 peptides. Intensity of serum binding in ELISA to each overlapping peptide covering the indicated regions of gp120 is shown as heatmap. In each panel stronger binding is represented as darker color.

Breadth of neutralizing antibodies (NAbs) elicited by vaccination with recombinant trimeric gp14094UG018

Induction of Nabs in rabbits immunized with recombinant trimeric gp14094UG018 was evaluated in the TZM-bl neutralization assay against a panel of 7 HIV-1 isolates using IgG purified from sera collected at week 0, 12 and 14 (T0, T12 and T14 respectively). In particular, 3 viruses were clade B isolates (Bx08, SF162 and QH0692), 2 were clade C viruses (MW965 and 92BR025), 1 virus belonged to the 02_AG subtype (DJ263.8) and 1 virus was a clade A and C mosaic isolate (92RW009). The virus isolates MW965, 92Br025, DJ263.8, Bx08 and SF162 are known to be Tier 1 while the virus isolate 92RW009 and QH0692 are classified as Tier 2 isolates. A heatmap was generated based upon the 50% neutralization effect, indicating in red the neutralization results above 50% for each IgG concentration (Figure 7A).
Figure 7

Neutralization activity of sera from immunized rabbits. (A) Neutralization activity in TZM-bl cells obtained with serial dilutions of IgG (expressed as μg/ml) purified from immunized sera is shown as heatmap for each virus tested. Neutralization activity >50% and <50% is indicated in red and orange, respectively, whereas absence of neutralization activity is indicated in grey. Pre-bleed sera showed a negligible neutralization activity (<10%). (B) Correlation matrix between neutralization activity and peptide binding to envelope gp120 regions. (C) Neutralization activity of TriMab.

The overall results showed that the immune sera were able to mainly neutralize tier 1 viruses across clades and that this neutralization potency waned over time. Considering the T12 sera, 3 of the 5 Tier 1 virus isolates (SF162, Bx08 and MW965) were neutralized with an IC50 of 62.5 μg/ml by all sera and with an IC50 of 31.25 μg/ml by serum from the 49391 rabbit. The latter serum showed a neutralization activity also against DJ263.8 with an IC50 of 125 μg/ml (Figure 7A and Table 1), while no neutralization against 92Br025 could be detected. Tier 2 viruses were poorly or not neutralized in all cases.

Comparing the breadth and potency of neutralization, it is noteworthy that the best performing serum (49391) showed the strongest binding to the constant regions (p <0.05) and the lowest binding to the variable regions (V2 and V3) (p < 0.001), compared to the other sera. On the contrary, the least performing sera (49378 and 49384) show a significant binding to the constant regions, but the strongest binding to the V2 and V3 variable regions (p < 0.01) (Figure 7B).

Discussion

In this study we evaluated the immunogenicity of a soluble trimeric gp14094UG018 derived from a clade A HIV-1 isolate administrated subcutaneously to rabbits. All four immunized animals developed high titers of specific anti-gp140 antibodies against the homologous gp14094UG018 protein, as well as against heterologous envelope glycoproteins of clade B (gp120IIIB and gp120W61D), clade A (gp140UG037) and clade C (gp140ZM96) which present a significant sequence divergence compared to the gp14094UG018. The highest binding activity was observed against the two gp140 UG037 and ZM96 molecules, effect that could be due to the highest sequence homology to the gp140 94UG018 molecule (UG037) and/or the presence of the ectodomain of gp41 which is substantially conserved across the clades (UG037 and ZM96). Overall, these results indicate that although rabbits were immunized with a clade A derived gp140, this immunization was able to elicit a binding activity to cross-clade Env antigens. This result was supported by the relevant sequence homology between the different Env molecules, especially in the constant regions.

All immunized animals’ sera were able to bind the C1, C2 and C5 regions with high affinity and the V2, V3 and V5 regions with lower affinity, presenting a direct correlation with the percentage of divergence between the gp14094UG018 immunogen and the peptides used as target in the binding assay. However, the differential binding efficacy to the same peptide by different sera is highly suggestive of distinct patterns of immune response elicited by the same gp14094UG018 in outbred animals, indicating that the specificity of individual responses to the same vaccine antigen is not totally predictable. A more in-depth epitope mapping analysis performed with individual epitopes covering the entire length of gp120 from the C1 to the C5 region confirmed the potency ranking observed with the peptide pools but showed that, for each region, specific peptides are recognized more efficiently than others. This further supports the concept that the immunogen is able to elicit distinct patterns of Abs focused on different epitopes. Further analysis have been planned to be conducted using peptides based on the sequence of 94UG018 protein, to verify whether additional Ab specificities are identified in immune sera elicited by the gp14094UG018.

Sera from animals immunized with gp14094UG018 showed a >50% neutralization efficacy against 3 out of 5 Tier 1 pseudoviruses, whereas poor or no neutralization was observed against Tier 2 pseudoviruses.

This soluble trimeric clade A gp14094UG018 was able to induce a cross-clade neutralizing activity as demonstrated by the ability of the immune sera to neutralize Tier 1 pseudoviruses of different clades. Although the neutralization activity was limited to Tier 1 pseudoviruses, this result is in agreement with other immunogenicity studies performed with soluble HIV Envelope proteins of different clades with our data even suggesting improved neutralization in several instances [3942]. Moreover, it is prospected to re-evaluate the breadth of neutralization activity in A3R5 cells, which have been recently shown to be more sensitive to neutralization than the TZM-bl used in the present study [43]. In particular, serum from the 49391 rabbit was the only one to show neutralization activity against four out of five Tier 1 with the highest activity and also to show very limited neutralization activity, although lower than the 50% threshold, against two of the Tier 2 pseudoviruses. This broader neutralization activity does not seem to be attributable to antibodies to the V3 domain, as serum from the 49391 rabbit, compared to the other sera, presents the weakest binding efficacy to V3 epitopes, considered both as pool or individual peptides. This observation is in contrast to previously reported data showing that broadly neutralizing activity, in sera from animals immunized with trimeric Clade A envelope molecules, is mostly associated to antibodies directed toward the envelope variable regions (V1, V2, V3) [44]. Interestingly, our gp14094UG018 seems to divert the immune response from variable region of envelope molecules. Further analyses will need to be performed since these results could be due to the high sequence divergence between the gp14094UG018 and the gp120IIIB peptides used in our assay.

Of note, all four immunized sera are characterized by a stronger binding efficacy to epitopes of the constant and V5 regions, which are known to be involved in the CD4bs of the HIV gp120 and are targets of few broadly cross-clade neutralizing monoclonal antibodies (bnAbs) [11, 45, 46]. The poor or absent binding to C3 epitopes is most likely due to the extremely high divergence (>60%) of the C3 region sequence in the gp14094UG018 which may severely affect the Ab-epitope recognition. Nevertheless, the strong binding to epitopes covering regions involved in the CD4bs may possibly suggest that such antibodies may play a relevant role in the neutralization activity of immunized sera, although the broadness of their activity is confined only to Tier 1 isolates. Indeed, the observed strong binding to C1 epitopes may possibly play a role in the insufficient pattern of neutralization, given that antibodies binding to C1-C4 domains have been reported to compete with CD4bs broadly cross-neutralizing antibodies for binding [47]. Moreover, Abs specific to the C1 region have been reported to be associated with induction of antibody-dependent cellular cytotoxicity (ADCC) [48], which plays a role in protection from HIV infection and disease progression, as shown also in the RV144 Thai vaccine trial [4951]. Overall, the results of the present study highlight that trimeric clade A gp14094UG018 is a very effective immunogen capable of inducing significant cross-clade humoral immune responses in the rabbit model. The gp120 epitope mapping provides potential relevant insights to clarify the neutralization activity of the elicited immune sera confined to Tier 1 isolates. According to these observations, possible structural modifications of the clade A gp14094UG018 can be envisaged (i.e. deletion of the C1 region) to improve the breadth of the neutralization activity.

Our results provide a rationale for the design and evaluation of immunogens to be used in HIV vaccine strategies. In particular, clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV immunization regimen.

Declarations

Acknowledgement

Work supported by EC-FP7-grant NGIN_201433.

Authors’ Affiliations

(1)
Molecular Biology and Viral Oncogenesis Unit, Department of Experimental Oncology, Istituto Nazionale per lo Studio e la Cura dei Tumori “Fondazione Pascale” - IRCCS
(2)
Human Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford University
(3)
Virology Unit, Institute of Tropical Medicine
(4)
Department of Laboratory Medicine, University of Lund
(5)
Statens Serum Institut
(6)
National Institute for Biological Standards and Control
(7)
San Raffaele Scientific Institute

References

  1. Phillips RE, Rowland-Jones S, Nixon DF, Gotch FM, Edwards JP, Ogunlesi AO, Elvin JG, Rothbard JA, Bangham CR, Rizza CR: Human immunodeficiency virus genetic variation that can escape cytotoxic T cell recognition. Nature. 1991, 354: 453-459. 10.1038/354453a0.View ArticlePubMedGoogle Scholar
  2. Richman DD, Wrin T, Little SJ, Petropoulos CJ: Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc Natl Acad Sci USA. 2003, 100: 4144-4149. 10.1073/pnas.0630530100.PubMed CentralView ArticlePubMedGoogle Scholar
  3. Wei X, Decker JM, Wang S, Hui H, Kappes JC, Wu X, Salazar-Gonzalez JF, Salazar MG, Kilby JM, Saag MS, Komarova NL, Nowak MA, Hahn BH, Kwong PD, Shaw GM: Antibody neutralization and escape by HIV-1. Nature. 2003, 422: 307-312. 10.1038/nature01470.View ArticlePubMedGoogle Scholar
  4. Overbaugh J, Morris L: The Antibody Response against HIV-1. Cold Spring Harb Perspect Med. 2012, 2: a007039-10.1101/cshperspect.a007039.PubMed CentralView ArticlePubMedGoogle Scholar
  5. Burton DR, Pyati J, Koduri R, Sharp SJ, Thornton GB, Parren PW, Sawyer LS, Hendry RM, Dunlop N, Nara PL: Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science. 1994, 266: 1024-1027. 10.1126/science.7973652.View ArticlePubMedGoogle Scholar
  6. Walker LM, Phogat SK, Chan-Hui PY, Wagner D, Phung P, Goss JL, Wrin T, Simek MD, Fling S, Mitcham JL, Lehrman JK, Priddy FH, Olsen OA, Frey SM, Hammond PW, Kaminsky S, Zamb T, Moyle M, Koff WC, Poignard P, Burton DR: Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science. 2009, 326: 285-289. 10.1126/science.1178746.PubMed CentralView ArticlePubMedGoogle Scholar
  7. Pantophlet R, Wrin T, Cavacini LA, Robinson JE, Burton DR: Neutralizing activity of antibodies to the V3 loop region of HIV-1 gp120 relative to their epitope fine specificity. Virology. 2008, 381: 251-260. 10.1016/j.virol.2008.08.032.PubMed CentralView ArticlePubMedGoogle Scholar
  8. Trkola A, Purtscher M, Muster T, Ballaun C, Buchacher A, Sullivan N, Srinivasan K, Sodroski JG, Moore JP, Katinger H: Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of Human Immunodeficiency Virus type 1. J Virol. 1996, 70: 1100-1108.PubMed CentralPubMedGoogle Scholar
  9. Muster T, Steindl F, Purtscher M, Trkola A, Klima A, Himmler G, Ruker F, Katinger H: A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J Virol. 1993, 67: 6642-6647.PubMed CentralPubMedGoogle Scholar
  10. Stiegler G, Kunert R, Purtscher M, Wolbank S, Voglauer R, Steindl F, Katinger H: A potent cross-clade neutralizing human monoclonal antibody against a novel epitope on gp41 of human immunodeficiency virus type 1. AIDS Res Hum Retroviruses. 2001, 17: 1757-1765. 10.1089/08892220152741450.View ArticlePubMedGoogle Scholar
  11. Wu X, Yang ZY, Li Y, Hogerkorp CM, Schief WR, Seaman MS, Zhou T, Schmidt SD, Wu L, Xu L, Longo NS, McKee K, O’Dell S, Louder MK, Wycuff DL, Feng Y, Nason M, Doria-Rose N, Connors M, Kwong PD, Roederer M, Wyatt RT, Nabel GJ, Mascola JR: Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science. 2010, 329: 856-861. 10.1126/science.1187659.PubMed CentralView ArticlePubMedGoogle Scholar
  12. Corti D, Langedijk JP, Hinz A, Seaman MS, Vanzetta F, Fernandez-Rodriguez BM, Silacci C, Pinna D, Jarrossay D, Balla-Jhagjhoorsingh S, Willems B, Zekveld MJ, Dreja H, O’Sullivan E, Pade C, Orkin C, Jeffs SA, Montefiori DC, Davis D, Weissenhorn W, McKnight A, Heeney JL, Sallusto F, Sattentau QJ, Weiss RA, Lanzavecchia A: Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals. PLoS One. 2010, 5: e8805-10.1371/journal.pone.0008805.PubMed CentralView ArticlePubMedGoogle Scholar
  13. Gorny MK, Williams C, Volsky B, Revesz K, Wang XH, Burda S, Kimura T, Konings FA, Nadas A, Anyangwe CA, Nyambi P, Krachmarov C, Pinter A, Zolla-Pazner S: Cross-clade neutralizing activity of human anti-V3 monoclonal antibodies derived from the cells of individuals infected with non-B clades of human immunodeficiency virus type 1. J Virol. 2006, 80: 6865-6872. 10.1128/JVI.02202-05.PubMed CentralView ArticlePubMedGoogle Scholar
  14. Bell CH, Pantophlet R, Schiefner A, Cavacini LA, Stanfield RL, Burton DR, Wilson IA: Structure of antibody F425-B4e8 in complex with a V3 peptide reveals a new binding mode for HIV-1 neutralization. J Mol Biol. 2008, 375: 969-978. 10.1016/j.jmb.2007.11.013.PubMed CentralView ArticlePubMedGoogle Scholar
  15. Beddows S, Schulke N, Kirschner M, Barnes K, Franti M, Michael E, Ketas T, Sanders RW, Maddon PJ, Olson WC, Moore JP: Evaluating the immunogenicity of a disulfide-stabilized, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. J Virol. 2005, 79: 8812-8827. 10.1128/JVI.79.14.8812-8827.2005.PubMed CentralView ArticlePubMedGoogle Scholar
  16. Beddows S, Franti M, Dey AK, Kirschner M, Iyer SP, Fisch DC, Ketas T, Yuste E, Desrosiers RC, Klasse PJ, Maddon PJ, Olson WC, Moore JP: A comparative immunogenicity study in rabbits of disulfide-stabilized, proteolytically cleaved, soluble trimeric human immunodeficiency virus type 1 gp140, trimeric cleavage-defective gp140 and monomeric gp120. Virology. 2007, 360: 329-340. 10.1016/j.virol.2006.10.032.View ArticlePubMedGoogle Scholar
  17. Kang YK, Andjelic S, Binley JM, Crooks ET, Franti M, Iyer SP, Donovan GP, Dey AK, Zhu P, Roux KH, Durso RJ, Parsons TF, Maddon PJ, Moore JP, Olson WC: Structural and immunogenicity studies of a cleaved, stabilized envelope trimer derived from subtype A HIV-1. Vaccine. 2009, 27: 5120-5132. 10.1016/j.vaccine.2009.06.037.View ArticlePubMedGoogle Scholar
  18. Tagliamonte M, Visciano ML, Tornesello ML, De SA, Buonaguro FM, Buonaguro L: HIV-Gag VLPs presenting trimeric HIV-1 gp140 spikes constitutively expressed in stable double transfected insect cell line. Vaccine. 2011, 29: 4913-4922. 10.1016/j.vaccine.2011.05.004.View ArticlePubMedGoogle Scholar
  19. Visciano ML, Diomede L, Tagliamonte M, Tornesello ML, Asti V, Bomsel M, Buonaguro FM, Lopalco L, Buonaguro L: Generation of HIV-1 Virus-Like Particles expressing different HIV-1 glycoproteins. Vaccine. 2011, 29: 4903-4912. 10.1016/j.vaccine.2011.05.005.View ArticlePubMedGoogle Scholar
  20. Eckert DM, Kim PS: Mechanisms of viral membrane fusion and its inhibition. Annu Rev Biochem. 2001, 70: 777-810. 10.1146/annurev.biochem.70.1.777.View ArticlePubMedGoogle Scholar
  21. Poignard P, Saphire EO, Parren PW, Burton DR: gp120: Biologic aspects of structural features. Annu Rev Immunol. 2001, 19: 253-274. 10.1146/annurev.immunol.19.1.253.View ArticlePubMedGoogle Scholar
  22. Wyatt R, Sodroski J: The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science. 1998, 280: 1884-1888. 10.1126/science.280.5371.1884.View ArticlePubMedGoogle Scholar
  23. Earl PL, Doms RW, Moss B: Oligomeric structure of the human immunodeficiency virus type 1 envelope glycoprotein. Proc Natl Acad Sci USA. 1990, 87: 648-652. 10.1073/pnas.87.2.648.PubMed CentralView ArticlePubMedGoogle Scholar
  24. Chen B, Zhou G, Kim M, Chishti Y, Hussey RE, Ely B, Skehel JJ, Reinherz EL, Harrison SC, Wiley DC: Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160. J Biol Chem. 2000, 275: 34946-34953. 10.1074/jbc.M004905200.View ArticlePubMedGoogle Scholar
  25. Earl PL, Sugiura W, Montefiori DC, Broder CC, Lee SA, Wild C, Lifson J, Moss B: Immunogenicity and protective efficacy of oligomeric human immunodeficiency virus type 1 gp140. J Virol. 2001, 75: 645-653. 10.1128/JVI.75.2.645-653.2001.PubMed CentralView ArticlePubMedGoogle Scholar
  26. Yang X, Lee J, Mahony EM, Kwong PD, Wyatt R, Sodroski J: Highly stable trimers formed by human immunodeficiency virus type 1 envelope glycoproteins fused with the trimeric motif of T4 bacteriophage fibritin. J Virol. 2002, 76: 4634-4642. 10.1128/JVI.76.9.4634-4642.2002.PubMed CentralView ArticlePubMedGoogle Scholar
  27. Kovacs JM, Nkolola JP, Peng H, Cheung A, Perry J, Miller CA, Seaman MS, Barouch DH, Chen B: HIV-1 envelope trimer elicits more potent neutralizing antibody responses than monomeric gp120. Proc Natl Acad Sci USA. 2012, 109: 12111-12116. 10.1073/pnas.1204533109.PubMed CentralView ArticlePubMedGoogle Scholar
  28. Binley JM, Sanders RW, Clas B, Schuelke N, Master A, Guo Y, Kajumo F, Anselma DJ, Maddon PJ, Olson WC, Moore JP: A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J Virol. 2000, 74: 627-643. 10.1128/JVI.74.2.627-643.2000.PubMed CentralView ArticlePubMedGoogle Scholar
  29. Sanders RW, Vesanen M, Schuelke N, Master A, Schiffner L, Kalyanaraman R, Paluch M, Berkhout B, Maddon PJ, Olson WC, Lu M, Moore JP: Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. J Virol. 2002, 76: 8875-8889. 10.1128/JVI.76.17.8875-8889.2002.PubMed CentralView ArticlePubMedGoogle Scholar
  30. Buonaguro L, DelGaudio E, Monaco M, Greco D, Corti P, Beth-Giraldo E, Buonaguro FM, Giraldo G: Heteroduplex mobility assay and phylogenetic analysis of V3 region sequences of HIV 1 isolates from Gulu - Northern Uganda. J Virol. 1995, 69: 7971-7981.PubMed CentralPubMedGoogle Scholar
  31. Buonaguro L, Buonaguro FM, Russo F, Tornesello ML, Beth-Giraldo E, Wagner R, Wolf H, Giraldo G: A novel gp120 sequence from an HIV-1 isolate of the A clade identified in North Uganda. AIDS Res Hum Retroviruses. 1998, 14: 1287-1289. 10.1089/aid.1998.14.1287.View ArticlePubMedGoogle Scholar
  32. Buonaguro L, Racioppi L, Tornesello ML, Arra C, Visciano ML, Biryahwaho B, Sempala SDK, Giraldo G, Buonaguro FM: Induction of neutralizing antibodies and CTLs in Balb/c mice immunized with Virus-like Particles presenting a gp120 molecule from a HIV-1 isolate of clade A (HIV-VLPAs). Antiviral Res. 2002, 54: 189-201. 10.1016/S0166-3542(02)00004-9.View ArticlePubMedGoogle Scholar
  33. Buonaguro L, Visciano ML, Tornesello ML, Tagliamonte M, Biryahwaho B, Buonaguro FM: Induction of systemic and mucosal cross-clade neutralizing antibodies in BALB/c mice immunized with human immunodeficiency virus type 1 clade A virus-like particles administered by different routes of inoculation. J Virol. 2005, 79: 7059-7067. 10.1128/JVI.79.11.7059-7067.2005.PubMed CentralView ArticlePubMedGoogle Scholar
  34. Buonaguro L, Devito C, Tornesello ML, Schroder U, Wahren B, Hinkula J, Buonaguro FM: DNA-VLP prime-boost intra-nasal immunization induces cellular and humoral anti-HIV-1 systemic and mucosal immunity with cross-clade neutralizing activity. Vaccine. 2007, 25: 5968-5977. 10.1016/j.vaccine.2007.05.052.View ArticlePubMedGoogle Scholar
  35. Buonaguro L, Tagliamonte M, Visciano ML, Andersen H, Lewis M, Pal R, Tornesello ML, Schroeder U, Hinkula J, Wahren B, Buonaguro FM: Immunogenicity of HIV virus-like particles in rhesus macaques by intranasal administration. Clin Vaccine Immunol. 2012, 19: 970-973. 10.1128/CVI.00068-12.PubMed CentralView ArticlePubMedGoogle Scholar
  36. Agger EM, Rosenkrands I, Hansen J, Brahimi K, Vandahl BS, Aagaard C, Werninghaus K, Kirschning C, Lang R, Christensen D, Theisen M, Follmann F, Andersen P: Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological requirements. PLoS One. 2008, 3: e3116-10.1371/journal.pone.0003116.PubMed CentralView ArticlePubMedGoogle Scholar
  37. Li M, Gao F, Mascola JR, Stamatatos L, Polonis VR, Koutsoukos M, Voss G, Goepfert P, Gilbert P, Greene KM, Bilska M, Kothe DL, Salazar-Gonzalez JF, Wei X, Decker JM, Hahn BH, Montefiori DC: Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol. 2005, 79: 10108-10125. 10.1128/JVI.79.16.10108-10125.2005.PubMed CentralView ArticlePubMedGoogle Scholar
  38. Jiang X, Burke V, Totrov M, Williams C, Cardozo T, Gorny MK, Zolla-Pazner S, Kong XP: Conserved structural elements in the V3 crown of HIV-1 gp120. Nat Struct Mol Biol. 2010, 17: 955-961. 10.1038/nsmb.1861.View ArticlePubMedGoogle Scholar
  39. Kraft Z, Strouss K, Sutton WF, Cleveland B, Tso FY, Polacino P, Overbaugh J, Hu SL, Stamatatos L: Characterization of neutralizing antibody responses elicited by clade A envelope immunogens derived from early transmitted viruses. J Virol. 2008, 82: 5912-5921. 10.1128/JVI.00389-08.PubMed CentralView ArticlePubMedGoogle Scholar
  40. Zhang PF, Cham F, Dong M, Choudhary A, Bouma P, Zhang Z, Shao Y, Feng YR, Wang L, Mathy N, Voss G, Broder CC, Quinnan GV: Extensively cross-reactive anti-HIV-1 neutralizing antibodies induced by gp140 immunization. Proc Natl Acad Sci USA. 2007, 104: 10193-10198. 10.1073/pnas.0608635104.PubMed CentralView ArticlePubMedGoogle Scholar
  41. Lian Y, Srivastava I, Gomez-Roman VR, Zur MJ, Sun Y, Kan E, Hilt S, Engelbrecht S, Himathongkham S, Luciw PA, Otten G, Ulmer JB, Donnelly JJ, Rabussay D, Montefiori D, Van Rensburg EJ, Barnett SW: Evaluation of envelope vaccines derived from the South African subtype C human immunodeficiency virus type 1 TV1 strain. J Virol. 2005, 79: 13338-13349. 10.1128/JVI.79.21.13338-13349.2005.PubMed CentralView ArticlePubMedGoogle Scholar
  42. Sellhorn G, Kraft Z, Caldwell Z, Ellingson K, Mineart C, Seaman MS, Montefiori DC, Lagerquist E, Stamatatos L: Engineering, expression, purification, and characterization of stable clade A/B recombinant soluble heterotrimeric gp140 proteins. J Virol. 2012, 86: 128-142. 10.1128/JVI.06363-11.PubMed CentralView ArticlePubMedGoogle Scholar
  43. Montefiori DC, Karnasuta C, Huang Y, Ahmed H, Gilbert P, De Souza MS, McLinden R, Tovanabutra S, Laurence-Chenine A, Sanders-Buell E, Moody MA, Bonsignori M, Ochsenbauer C, Kappes J, Tang H, Greene K, Gao H, LaBranche CC, Andrews C, Polonis VR, Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Self SG, Berman PW, Francis D, Sinangil F, Lee C, Tartaglia J: Magnitude and breadth of the neutralizing antibody response in the RV144 and Vax003 HIV-1 vaccine efficacy trials. J Infect Dis. 2012, 206: 431-441. 10.1093/infdis/jis367.PubMed CentralView ArticlePubMedGoogle Scholar
  44. Nkolola JP, Peng H, Settembre EC, Freeman M, Grandpre LE, Devoy C, Lynch DM, La PA, Simmons NL, Bradley R, Montefiori DC, Seaman MS, Chen B, Barouch DH: Breadth of neutralizing antibodies elicited by stable, homogeneous clade A and clade C HIV-1 gp140 envelope trimers in guinea pigs. J Virol. 2010, 84: 3270-3279. 10.1128/JVI.02252-09.PubMed CentralView ArticlePubMedGoogle Scholar
  45. Wu X, Zhou T, Zhu J, Zhang B, Georgiev I, Wang C, Chen X, Longo NS, Louder M, McKee K, O’Dell S, Perfetto S, Schmidt SD, Shi W, Wu L, Yang Y, Yang ZY, Yang Z, Zhang Z, Bonsignori M, Crump JA, Kapiga SH, Sam NE, Haynes BF, Simek M, Burton DR, Koff WC, Doria-Rose NA, Connors M, Mullikin JC: Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing. Science. 2011, 333: 1593-1602. 10.1126/science.1207532.PubMed CentralView ArticlePubMedGoogle Scholar
  46. Scheid JF, Mouquet H, Ueberheide B, Diskin R, Klein F, Oliveira TY, Pietzsch J, Fenyo D, Abadir A, Velinzon K, Hurley A, Myung S, Boulad F, Poignard P, Burton DR, Pereyra F, Ho DD, Walker BD, Seaman MS, Bjorkman PJ, Chait BT, Nussenzweig MC: Sequence and structural convergence of broad and potent HIV antibodies that mimic CD4 binding. Science. 2011, 333: 1633-1637. 10.1126/science.1207227.PubMed CentralView ArticlePubMedGoogle Scholar
  47. Moore JP, Sodroski J: Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein. J Virol. 1996, 70: 1863-1872.PubMed CentralPubMedGoogle Scholar
  48. Ampol S, Pattanapanyasat K, Sutthent R, Permpikul P, Kantakamalakul W: Comprehensive Investigation of Common Antibody-Dependent Cell-Mediated Cytotoxicity Antibody Epitopes of HIV-1 CRF01_AE gp120. AIDS Res Hum Retroviruses. 2012, 28: 1250-1258. 10.1089/aid.2011.0346.View ArticlePubMedGoogle Scholar
  49. Brocca-Cofano E, McKinnon K, Demberg T, Venzon D, Hidajat R, Xiao P, Tabuit-Test M, Patterson LJ, Robert-Guroff M: Vaccine-elicited SIV and HIV envelope-specific IgA and IgG memory B cells in rhesus macaque peripheral blood correlate with functional antibody responses and reduced viremia. Vaccine. 2011, 29: 3310-3319. 10.1016/j.vaccine.2011.02.066.PubMed CentralView ArticlePubMedGoogle Scholar
  50. Lambotte O, Ferrari G, Moog C, Yates NL, Liao HX, Parks RJ, Hicks CB, Owzar K, Tomaras GD, Montefiori DC, Haynes BF, Delfraissy JF: Heterogeneous neutralizing antibody and antibody-dependent cell cytotoxicity responses in HIV-1 elite controllers. AIDS. 2009, 23: 897-906. 10.1097/QAD.0b013e328329f97d.PubMed CentralView ArticlePubMedGoogle Scholar
  51. Wren L, Kent SJ: HIV Vaccine efficacy trial: glimmers of hope and the potential role of antibody-dependent cellular cytotoxicity. Hum Vaccin. 2011, 7: 466-473. 10.4161/hv.7.4.14123.View ArticlePubMedGoogle Scholar
  52. Abagyan R, Totrov M, Kuznetsov D: ICM—A new method for protein modeling and design: Applications to docking and structure prediction from the distorted native conformation. J Comp Chem. 1994, 15: 488-506. 10.1002/jcc.540150503.View ArticleGoogle Scholar

Copyright

© Visciano et al.; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Advertisement