mAb 2F5 Research Articles

The TZM-bl Reporter Cell Line Expresses Kynureninase That Can Neutralize 2F5-like Antibodies in the HIV-1 Neutralization Assay.

Induction of broadly neutralizing antibodies targeting ectodomain of the transmembrane (TM) glycoprotein gp41 HIV-1 provides a basis for the development of a universal anti-viral vaccine. The HeLa cell-derived TZM-bl reporter cell line is widely used for the estimation of lentiviruses neutralization by immune sera. The cell line is highly permissive to infection by most strains of HIV, SIV, and SHIV. Here we demonstrated that TZM-bl cells express a 48 kDa non-glycosylated protein (p48) recognized by broadly neutralizing monoclonal antibody (mAb) 2F5 targeting the ELDKWA (aa 669–674) epitope of gp41TM of HIV-1. A significant amount of p48 was found in the cell supernatant. The protein was identified as human kynureninase (KYNU), which has the ELDKWA epitope. The protein is further called “p48 KYNU”. The HIV-1 neutralization by mAb 2F5 and 4E10 in the presence of p48KYNU was tested on Jurkat and TZM-bl cells. It was demonstrated that p48KYNU reduces neutralization by 2F5-like antibodies, but it has almost no effect on mAb 4E10. Therefore, p48KYNU can attenuate HIV-1 neutralization by 2F5-like antibodies and hence create false-negative results. Thus, previously tested immune sera that recognized the ELDKWA-epitope and demonstrated a “weak neutralization” of HIV-1 in TZM-bl assay should be reevaluated.

Germinality does not necessarily define mAb expression and thermal stability

The production potential of recombinant monoclonal antibody (mAb) expressing cell lines depends, among other factors, on the intrinsic antibody structure determined by the amino acid sequence. In this study, we investigated the influence of somatic mutations in the V(D)J sequence of four individual, mature model mAbs on the expression potential. Therefore, we defined four couples, each consisting of one naturally occurring mAb (2G12, Ustekinumab, 4B3, and 2F5) and the corresponding germline-derived cognate mAb (353/11, 554/12, 136/63, and 236/14). For all eight mAb variants, recombinant Chinese hamster ovary (CHO) cell lines were developed with mAbs expressed from a defined chromosomal locus. The presented workflow investigates critical parameters including productivity, intra- and extracellular product profile, XBP1 splicing, thermal stability, and in silico hydrophobicity. Significant differences in productivity were even observed between the germline-derived mAbs which did not undergo somatic mutagenesis. Accordingly, back-to-germline mutations of mature mAbs are not necessarily reflecting improved expression and stability but indicate opportunities and limits of mAb engineering. From our studies, we conclude that germinalization represents a potential to improve mAb properties depending on the antibody’s germline family, highlighting the fact that mAbs should be treated individually.

Identification of a HIV Gp41-Specific Human Monoclonal Antibody With Potent Antibody-Dependent Cellular Cytotoxicity.

Antibody-Dependent Cellular Cytotoxicity (ADCC) is a major mechanism of protection against viral infections in vivo. Identification of HIV-1-specific monoclonal antibodies (mAbs) with potent ADCC activity may help develop an effective HIV-1 vaccine. In present study, we isolated such human mAb, designated E10, from an HIV-1-infected patient sample by single B cell sorting and single cell PCR. E10 bound to gp140 trimer and linear peptides derived from gp41 membrane proximal external region (MPER). E10 epitope (QEKNEQELLEL) overlapped with mAb 2F5 epitope. However, E10 differentiated from 2F5 in neutralization breadth and potency, as well as ADCC activity. E10 showed low neutralization activity and narrow spectrum of neutralization compared to 2F5, but it mediated higher ADCC activity than 2F5 at low antibody concentration. Fine mapping of E10 epitope may potentiate MPER-based subunit vaccine development.

Lessons learned from merging wet lab experiments with molecular simulation to improve mAb humanization.

Humanized monoclonal antibodies (mAbs) are among the most promising modern therapeutics, but defined engineering strategies are still not available. Antibody humanization often leads to a loss of affinity, as it is the case for our model antibody Ab2/3H6 (PDB entry 3BQU). Identifying appropriate back-to-mouse mutations is needed to restore binding affinity, but highly challenging. In order to get more insight, we have applied molecular dynamics simulations and correlated them to antibody binding and expression in wet lab experiments. In this study, we discuss six mAb variants and investigate a tyrosine conglomeration, an isopolar substitution and the improvement of antibody binding towards wildtype affinity. In the 3D structure of the mouse wildtype, residue R94h is surrounded by three tyrosines which form a so-called ‘tyrosine cage’. We demonstrate that the tyrosine cage has a supporting function for the CDRh3 loop conformation. The isopolar substitution is not able to mimic the function appropriately. Finally, we show that additional light chain mutations can restore binding to wildtype-comparable level, and also improve the expression of the mAb significantly. We conclude that the variable light chain of Ab2/3H6 is of underestimated importance for the interaction with its antigen mAb 2F5.

Optimization of the EC26-2A4 Epitope in the gp41 Membrane Proximal External Region Targeted by Neutralizing Antibodies from an Elite Controller.

The analysis of patient derived HIV neutralizing antibodies (nAbs) and their target epitopes in the viral envelope (Env) protein provides important basic information for vaccine design. In this study we optimized an epitope, EC26-2A4, that is targeted by neutralizing antibodies from an elite controller (EC26) and localizes in the membrane-proximal external region from the gp41 transmembrane protein. Due to its overlap with the epitope of the first generation broadly neutralizing monoclonal Ab (mAb) 2F5 associated with autoreactivity, we first defined the minimal core epitope reacting with antibodies from EC26 plasma, but not with mAb 2F5. The optimized minimal epitope, EC26-2A4ΔM, was able to induce neutralizing antibodies in vaccinated mice. We further analyzed the frequency of antibodies against the EC26-2A4ΔM peptide in HIV-positive patient sera from a treated cohort and an untreated long-term nonprogressor (LTNP) cohort. Interestingly, 27% of the LTNP sera reacted with the peptide, whereas only 9% showed reactivity in the treated cohort. Although there was no association between the presence of antibodies against the EC26-2A4ΔM epitope and viral load or CD4 count in these patients, the CD4 nadir in the treated cohort was higher in patients positive for EC26-2A4ΔM antibodies, in particular in patients having such antibodies at an early and a late timepoint after infection.

Structural insights into the IgE mediated responses induced by the allergens Hev b 8 and Zea m 12 in their dimeric forms.

Oligomerization of allergens plays an important role in IgE-mediated reactions, as effective crosslinking of IgE- FcεRI complexes on the cell membrane is dependent on the number of exposed B-cell epitopes in a single allergen molecule or on the occurrence of identical epitopes in a symmetrical arrangement. Few studies have attempted to experimentally demonstrate the connection between allergen dimerization and the ability to trigger allergic reactions. Here we studied plant allergenic profilins rHev b 8 (rubber tree) and rZea m 12 (maize) because they represent an important example of cross-reactivity in the latex-pollen-food syndrome. Both allergens in their monomeric and dimeric states were isolated and characterized by exclusion chromatography and mass spectrometry and were used in immunological in vitro experiments. Their crystal structures were solved, and for Hev b 8 a disulfide-linked homodimer was found. Comparing the structures we established that the longest loop is relevant for recognition by IgE antibodies, whereas the conserved regions are important for cross-reactivity. We produced a novel monoclonal murine IgE (mAb 2F5), specific for rHev b 8, which was useful to provide evidence that profilin dimerization considerably increases the IgE-mediated degranulation in rat basophilic leukemia cells.

Artificial polyepitope HIV-1 immunogen containing mimotope of 2F5 epitope.

Constructing a vaccine against HIV-1, able to induce production of broadly neutralizing antibodies, is crucial. We report here the selection and characterization of RDWSFDRWSLSEFWL peptide mimotope that binds specifically to bNAbs 2F5. The peptide mimotope was selected from 15-mer phage-displayed peptide library by using Mab 2F5 as the selecting agent. The most abundant RDWSFDRWSLSEFWL peptide was inserted into a carrier, an artificial polyepitope immunogen - TBI (T- and B-cell immunogen). TBI-2F5 polyepitope immunogen that includes the mimotope of 2F5 epitope was constructed. It was shown that sera of mice immunized with TBI-2F5 protein recognized TBI protein as well as RDWSFDRWSLSEFWL peptide. The capacity of sera of immunized mice to neutralize HIV-1 was demonstrated using subtype B env-pseudoviruses of HIV-1 QH0692.42 and PVO.4. Based on these results, we conclude that peptide mimotope of 2F5 epitope RDWSFDRWSLSEFWL can be an essential component for a successful HIV-vaccine.

F-109 Antigenicity of the human immunodeficiency virus envelope on virions in solution by fluorescence correlation spectroscopy

The envelope glycoproteins on HIV particles are structurally heterogeneous, making it difficult to equate antigenicity with neutralization sensitivity. Efforts to address this question typically employ immobilized reagents and indirect measures of antibody interactions, which can produce inexact antigenicity profiles. Towards this, we applied a single molecule based approach using fluorescence correlation spectroscopy (FCS) that uses fluctuations in fluorescent signals to measure diffusion and reaction kinetics of fluorescently-labeled anti-envelope monoclonal antibodies (MAbs) as they attach to virions. Our method allows monitoring antibody-virion binding interaction with all reactants continuously in solution. FCS binding profiles of anti-envelope antibodies were determined using different virus types. Anti-gp120 MAbs against the 2G12 or b12 epitopes marking functional envelope structures potently neutralized CCR5-tropic JRFL and BaL pseudotyped viruses and exhibited efficient virion binding in solution. MAbs against various CD4-induced (CD4i) epitopes considered hidden on functional envelope structures exhibited limited binding to these pseudoviruses and were not neutralizing. Anti- gp41 MAb 2F5 neutralized both pseudoviruses despite limited virion binding. Overall our experimental data suggest that virion antigenicity in solution is not the only factor that might determine neutralization sensitivity. A variety of mechanisms may converge to produce the overall neutralizing profile of a given HIV variant, depending on variables such as envelope genotype, coreceptor tropism, and virus production method.

Antigenic Properties of the HIV Envelope on Virions in Solution

The structural flexibility found in human immunodeficiency virus (HIV) envelope glycoproteins creates a complex relationship between antigenicity and sensitivity to antiviral antibodies. The study of this issue in the context of viral particles is particularly problematic as conventional virus capture approaches can perturb antigenicity profiles. Here, we employed a unique analytical system based on fluorescence correlation spectroscopy (FCS), which measures antibody-virion binding with all reactants continuously in solution. Panels of nine anti-envelope monoclonal antibodies (MAbs) and five virus types were used to connect antibody binding profiles with neutralizing activities. Anti-gp120 MAbs against the 2G12 or b12 epitope, which marks functional envelope structures, neutralized viruses expressing CCR5-tropic envelopes and exhibited efficient virion binding in solution. MAbs against CD4-induced (CD4i) epitopes considered hidden on functional envelope structures poorly bound these viruses and were not neutralizing. Anti-gp41 MAb 2F5 was neutralizing despite limited virion binding. Similar antigenicity patterns occurred on CXCR4-tropic viruses, except that anti-CD4i MAbs 17b and 19e were neutralizing despite little or no virion binding. Notably, anti-gp120 MAb PG9 and anti-gp41 MAb F240 bound to both CCR5-tropic and CXCR4-tropic viruses without exerting neutralizing activity. Differences in the virus production system altered the binding efficiencies of some antibodies but did not enhance antigenicity of aberrant gp120 structures. Of all viruses tested, only JRFL pseudoviruses showed a direct relationship between MAb binding efficiency and neutralizing potency. Collectively, these data indicate that the antigenic profiles of free HIV particles generally favor the exposure of functional over aberrant gp120 structures. However, the efficiency of virion-antibody interactions in solution inconsistently predicts neutralizing activity in vitro.

Chimeric Rhinoviruses Displaying MPER Epitopes Elicit Anti-HIV Neutralizing Responses

BackgroundThe development of an effective AIDS vaccine has been a formidable task, but remains a critical necessity. The well conserved membrane-proximal external region (MPER) of the HIV-1 gp41 glycoprotein is one of the crucial targets for AIDS vaccine development, as it has the necessary attribute of being able to elicit antibodies capable of neutralizing diverse isolates of HIV.Methodology/Principle FindingsGuided by X-ray crystallography, molecular modeling, combinatorial chemistry, and powerful selection techniques, we designed and produced six combinatorial libraries of chimeric human rhinoviruses (HRV) displaying the MPER epitopes corresponding to mAbs 2F5, 4E10, and/or Z13e1, connected to an immunogenic surface loop of HRV via linkers of varying lengths and sequences. Not all libraries led to viable chimeric viruses with the desired sequences, but the combinatorial approach allowed us to examine large numbers of MPER-displaying chimeras. Among the chimeras were five that elicited antibodies capable of significantly neutralizing HIV-1 pseudoviruses from at least three subtypes, in one case leading to neutralization of 10 pseudoviruses from all six subtypes tested.ConclusionsOptimization of these chimeras or closely related chimeras could conceivably lead to useful components of an effective AIDS vaccine. While the MPER of HIV may not be immunodominant in natural infection by HIV-1, its presence in a vaccine cocktail could provide critical breadth of protection.

A gp41 MPER-specific Llama VHH Requires a Hydrophobic CDR3 for Neutralization but not for Antigen Recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

Identification of a new epitope for HIV‐neutralizing antibodies in the gp41 membrane proximal external region by an Env‐tailored phage display library

HIV controllers are a valuable source for the identification of HIV-neutralizing antibodies, as chronic infection over decades allows extensive affinity maturation of antibodies for improved Ag recognition. We analyzed a small cohort of elite controllers (ECs) for HIV-neutralizing antibodies using a panel of standardized HIV-1 pseudovirions on TZM-bl cells. An HIV-1 Env-tailored phage display library was generated to select epitopes targeted by neutralizing antibodies in the EC26 plasma sample showing the broadest neutralizing activity. Selected Env fragments were mostly allocated to the membrane proximal external region of gp41. After preabsorbing the EC26 plasma with the selected phage EC26-2A4, we achieved 50% depletion of its neutralizing activity. Furthermore, antibodies affinity-purified with the EC26-2A4 epitope from EC26 plasma showed neutralizing activity, proving that the selected phage indeed contains an epitope targeted by neutralizing plasma antibodies. Epitope fine mapping of the purified plasma antibodies on peptide arrays identified a new epitope overlapping, but clearly distinct, from the prominent 2F5 epitope. Of note, the purified antibodies did not show autoreactivity with cardiolipin, whereas low reactivity with phosphatidylserine comparable to mAb 2F5 was observed. Thus, this new epitope represents a promising candidate for further analysis in view of HIV vaccine development.

Neutralization of porcine endogenous retrovirus by antibodies against the membrane-proximal external region of the transmembrane envelope protein

Immunization of different species including goats, rats, hamsters and guinea pigs with the recombinant ectodomain of the transmembrane envelope (TM) protein p15E of porcine endogenous retrovirus (PERV) has been shown to result in the production of virus-neutralizing antibodies. The sera recognize two groups of epitopes, one located in the fusion peptide-proximal region (FPPR) and the second in the membrane-proximal external region (MPER) of p15E. Most interestingly, the epitopes in the MPER are similar to epitopes in the TM protein gp41 of human immunodeficiency virus type 1 (HIV-1) recognized by mAbs 2F5 and 4E10, which broadly neutralize HIV-1. To study which epitope and which antibody population are involved in the process of neutralization of PERV, this study generated a new antiserum in a goat using an elongated ectodomain of p15E. The immune serum neutralized PERV at a higher titre and recognized broader epitopes in the FPPR and MPER of p15E. For the first time, antibody subpopulations were isolated from this serum using affinity chromatography with immobilized proteins and peptides corresponding to the FPPR and MPER of p15E. Only the affinity-purified antibodies specifically binding the MPER neutralized PERV, indicating that, as in the case of HIV-1, the MPER is an important target of neutralizing activity.

Functional characterization of the recombinant HIV-neutralizing monoclonal antibody 2F5 produced in maize seeds

Monoclonal antibodies (mAbs) that neutralize human immunodeficiency virus (HIV) can be used as microbicides to help prevent the spread of HIV in human populations. As an industry standard, HIV-neutralizing mAbs are produced as recombinant proteins in mammalian cells, but the high manufacturing costs and limited capacity reduce the ability of target populations in developing countries to gain access to these potentially life-saving medicines. Plants offer a more cost-effective and deployable production platform because they can be grown inexpensively and on a large scale in the region where the products are required. Here we show that the maize-derived HIV-neutralizing mAb 2F5 is assembled correctly in planta and binds to its antigen with the same affinity as 2F5 produced in mammalian cells. Although 2F5 has been produced at high levels in non-plant platforms, the yield in maize seeds is lower than previously achieved with another HIV-neutralizing mAb, 2G12. This suggests that the intrinsic properties of the antibody (e.g. sensitivity to specific proteases) and the environment provided by the production host (e.g. the relative abundance of different proteases, potential transgene silencing) may combine to limit the accumulation of some antibodies on a case-by-case basis.

Identification and characterization of a broadly cross-reactive HIV-1 human monoclonal antibody that binds to both gp120 and gp41.

Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs) mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs) by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER) of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s) of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics.

Short constrained peptides derived from phage display libraries as epitope models: the case of mAb 2F5

Background Since the monoclonal antibody 2F5 (mAb 2F5) was isolated in the early 90’s, its epitope have continued to be the focus of extensive investigations attempting to elucidate the mechanism by which impedes viral entry into host cells. Because the DKW-flanking amino acids are strongly conserved in viruses, it is not clear whether the DKW only satisfies the 2F5 epitope recognition demand. Methods We used phage display technology involving biopanning of a pIII-type 7-mer constrained peptide library (not screened in previous experiments with 2F5) for its epitope mimics. After peptides selection and widely characterization of several phage-peptide clones, some of them were used as immunogens. Polyclonal antibodies were evaluated as cell-cell fusion inhibitors of the CD4-Env complex interactions. Results We found that the specificity of recognition of the epitope depends on the structural context in which the cognate epitope sequence is presented. The antibody does not tolerate any replacements of the DKW-flanking epitope amino acids and binds exclusively to the (L)DKWA sequence provided by a 7-mer constrained peptide exposed by the M13 phage pIII protein. Additionally, immunization data supports the notion that the binding and neutralizing immunogenic structural features of the described epitope model do not coincide. Conclusion In this study, we show that when mAb 2F5 screens a pIIItype phage display 7-mer constrained peptide library for its epitope mimics, it demands an epitope sequence longer than DKW and does not tolerate substitutions in the epitope amino acid sequence as has been suggested in previous reports.

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 determinant for neutralization but not for antigen recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

Neutralization of Tier-2 Viruses and Epitope Profiling of Plasma Antibodies from Human Immunodeficiency Virus Type 1 Infected Donors from India

Broadly cross neutralizing antibodies (NAbs) are generated in a group of HIV-1 infected individuals during the natural infection, but little is known about their prevalence in patients infected with viral subtypes from different geographical regions. We tested here the neutralizing efficiency of plasma antibodies from 80 HIV-1 infected antiretroviral drug naive patients against a panel of subtype-B and C tier 2 viruses. We detected cross-neutralizing antibodies in approximately 19–27% of the plasma, however the subtype-C specific neutralization efficiency predominated (p = 0.004). The neutralizing activity was shown to be exclusively mediated by the immunoglobulin G (IgG) fraction in the representative plasma samples. Epitope mapping of three, the most cross-neutralizing plasma (CNP) AIIMS206, AIIMS239 and AIIMS249 with consensus-C overlapping envelope peptides revealed ten different binding specificities with only V3 and IDR being common. The V3 and IDR were highly antigenic regions but no correlation between their reciprocal Max50 binding titers and neutralization was observed. In addition, the neutralizing activity of CNP was not substantially reduced by V3 and gp41 peptides except a modest contribution of MPER peptide. The MPER was rarely recognized by plasma antibodies though antibody depletion and competition experiments demonstrated MPER dependent neutralization in two out of three CNP. Interestingly, the binding specificity of one of the CNP (AIIMS206) overlapped with broadly neutralizing mAb 2F5 epitope. Overall, the data suggest that, despite the low immunogenicity of HIV-1 MPER, the antibodies directed to this region may serve as crucial reagents for HIV-1 vaccine design.

The Influence of biotinylation of 2F5 antibody on peptide selection from the combinatorial phage library

The effect of biotinylation of monoclonal antibodies (MAbs) on the yield and repertoire of peptides selected by biopanning is discussed in the present study. A comparative analysis of peptides selected from a phage library using the biotinylated and nonbiotinylated MAb 2F5 was performed. The yield of the peptides homologous to the native epitope was observed to be 1.7 times higher for the biotinylated antibodies, whereas the use of the nonbiotinylated antibodies in the enzyme-linked immunoassay (ELISA) resulted in a greater binding capacity of the selected phages with MAb 2F5. It is notable that the phages, displaying peptides that have a fouror five-amino-acid sequence similarity with the native epitope, demonstrate the highest binding affinity to the antibodies. The phages exposing peptides with three-amino-acid sequence similarity exhibit various binding affinities. In summary, the results have revealed that a rational biopanning protocol should be chosen according to the aim of each particular study.

HIV-1 Virus-Like Particles Bearing Pure Env Trimers Expose Neutralizing Epitopes but Occlude Nonneutralizing Epitopes

Hypothetically, since native HIV-1 Env trimers are exclusively recognized by neutralizing antibodies, they might induce the neutralizing antibodies in a vaccine setting. This idea has not been evaluated due to the difficulty of separating trimers from nonfunctional Env (uncleaved gp160 and gp41 stumps). The latter are immunodominant and induce nonneutralizing antibodies. We previously showed that nonfunctional Env can be selectively cleared from virus-like particle (VLP) surfaces by enzyme digests (E. T. Crooks, T. Tong(,) K. Osawa, and J. M. Binley, J.Virol. 85:5825, 2011). Here, we investigated the effects of these digests on the antigenicity of VLPs and their sensitivity to neutralization. Before digestion, WT VLPs (bearing wild-type Env) and UNC VLPs (bearing uncleaved gp160) were recognized by various Env-specific monoclonal antibodies (MAbs), irrespective of their neutralizing activity, a result which is consistent with the presence of nonfunctional Env. After digestion, only neutralizing MAbs recognized WT VLPs, consistent with selective removal of nonfunctional Env (i.e., "trimer VLPs"). Digests eliminated the binding of all MAbs to UNC VLPs, again consistent with removal of nonfunctional Env. An exception was MAb 2F5, which weakly bound to digested UNC VLPs and bald VLPs (bearing no Env), perhaps due to lipid cross-reactivity. Trimer VLPs were infectious, and their neutralization sensitivity was largely comparable to that of undigested WT VLPs. However, they were ∼100-fold more sensitive to the MAbs 4E10 and Z13e1, suggesting increased exposure of the gp41 base. Importantly, a scatterplot analysis revealed a strong correlation between MAb binding and neutralization of trimer VLPs. This suggests that trimer VLPs bear essentially pure native trimer that should allow its unfettered evaluation in a vaccine setting.