Superinfection by discordant subtypes of HIV-1 does not enhance the neutralizing antibody response against autologous virus

Recent studies have demonstrated that both the potency and breadth of the humoral anti-HIV-1 immune response in generating neutralizing antibodies (nAbs) against heterologous viruses are significantly enhanced after superinfection by discordant HIV-1 subtypes, suggesting that repeated exposure of the immune system to highly diverse HIV-1 antigens can significantly improve anti-HIV-1 immunity. Thus, we investigated whether sequential plasma from these subjects superinfected with discordant HIV-1 subtypes, who exhibit broad nAbs against heterologous viruses, also neutralize their discordant early autologous viruses with increasing potency. Comparing the neutralization capacities of sequential plasma obtained before and after superinfection of 4 subjects to those of matched plasma obtained from 4 singly infected control subjects, no difference in the increase in neutralization capacity was observed between the two groups (p = 0.328). Overall, a higher increase in neutralization over time was detected in the singly infected patients (mean change in IC(50) titer from first to last plasma sample: 183.4) compared to the superinfected study subjects (mean change in IC(50) titer from first to last plasma sample: 66.5). Analysis of the Breadth-Potency Scores confirmed that there was no significant difference in the increase in superinfected and singly infected study subjects (p = 0.234). These studies suggest that while superinfection by discordant subtypes induces antibodies with enhanced neutralizing breadth and potency against heterologous viruses, the potency to neutralize their autologous viruses is not better than those seen in singly infected patients.     

Enhancement of alpha -helicity in the HIV-1 inhibitory peptide DP178 leads to an increased affinity for human monoclonal antibody 2F5 but does not elicit neutralizing responses in vitro. Implications for vaccine design

The synthetic peptide DP178, derived from the carboxyl-terminal heptad repeat region of human immunodeficiency virus type 1 GP41 protein is a potent inhibitor of viral-mediated fusion and contains the sequence ELDKWA, which constitutes the recognition epitope for the broadly neutralizing human monoclonal antibody 2F5. Efforts at eliciting a 2F5-like immune response by immunization with peptides or fusion proteins containing this sequence have not met with success, possibly because of incorrect structural presentation of the epitope. Although the structure of the carboxyl-terminal heptad repeat on the virion is not known, several recent reports have suggested a propensity for alpha-helical conformation. We have examined DP178 in the context of a model for optimized alpha-helices and show that the native sequence conforms poorly to the model. Solution conformation of DP178 was studied by circular dichroism and NMR spectroscopy and found to be predominantly random, consistent with previous reports. NMR mapping was used to show that the low percentage of alpha-helix present was localized to residues Glu(662) through Asn(671), a region encompassing the 2F5 epitope. Using NH(2)-terminal extensions derived from either GP41 or the yeast GCN4 leucine zipper dimerization domain, we designed peptide analogs in which the average helicity is significantly increased compared with DP178 and show that these peptides exhibit both a modest increase in affinity for 2F5 using a novel competitive solution-based binding assay and an increased ability to inhibit viral entry in a single-cycle infectivity model. Selected peptides were conjugated to carrier protein and used for guinea pig immunizations. High peptide-specific titers were achieved using these immunogens, but the resulting sera were incapable of viral neutralization. We discuss these findings in terms of structural and immunological considerations as to the utility of a 2F5-like response.     

Crystal structure of a cross-reaction complex between an anti-HIV-1 protease antibody and an HIV-2 protease peptide

The monoclonal antibody 1696, elicited by HIV-1 protease, inhibits the activity of both HIV-1 and HIV-2 proteases with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides derived from the N-terminal region of both proteases. The crystal structure of the recombinant single-chain Fv fragment of 1696 complexed with an N-terminal peptide from the HIV-2 protease has been determined at 1.88A resolution. Interactions of the peptide with scFv1696 are compared with the previously reported structure of scFv1696 in complex with the corresponding peptide from HIV-1 protease. The origin of cross-reactivity of mAb1696 with HIV proteases is discussed.     

An engineered lox sequence containing part of a long terminal repeat of HIV-1 permits Cre recombinase-mediated DNA excision.

In our previous report, one 34-bp sequence from a long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) clone, loxLTR-1, was proposed as a target site for site-specific excision by modified Cre recombinase. To support this suggestion, an engineered lox sequence, designated loxIL1, was made. This variant lox has the corresponding sequence of loxLTR-1 at the spacer region and the last two bases of inverted repeat sequence. Through in vitro recombination assay, loxIL1 also allowed the wild-type Cre to specifically recombine the sequence. An in vitro DNA binding experiment with mutants CreK244R and CreK244L revealed that lysine 244 of Cre plays an important role in interaction with the engineered lox. This result suggests that loxLTR-1 would be a candidate for antiviral strategy using site-specific recombinase.     

Gain of local structure in an amphipathic peptide does not require a specific tertiary framework

In this work, we studied how an amphipathic peptide of the surface of the globular protein thioredoxin, TRX94‐108, acquires a native‐like structure when it becomes involved in an apolar interaction network. We designed peptide variants where the tendency to form α‐helical conformation is modulated by replacing each of the leucine amino acid residues by an alanine. The induction of structure caused by sodium dodecyl sulfate (SDS) binding was studied by capillary zone electrophoresis, circular dichroism, DOSY‐NMR, and molecular dynamics simulations (MDS). In addition, we analyzed the strength of the interaction between a C18 RP‐HPLC matrix and the peptides. The results presented here reveal that (a) critical elements in the sequence of the wild‐type peptide stabilize a SDS/peptide supramolecular cluster; (b) the hydrophobic nature of the interaction between SDS molecules and the peptide constrains the ensemble of conformations; (c) nonspecific apolar surfaces are sufficient to stabilize peptide secondary structure. Remarkably, MDS shed light on a contact network formed by a limited number of SDS molecules that serves as a structural scaffold preserving the helical conformation of this module. This mechanism might prevail when a peptide with low helical propensity is involved in structure consolidation. We suggest that folding of peptides sharing this feature does not require a preformed tightly‐packed protein core. Thus, the formation of specific tertiary interactions would be the consequence of peptide folding and not its cause. In this scenario, folding might be thought of as a process that includes unspecific rounds of structure stabilization guiding the protein to the native state. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

An engineered construct of cFLIP provides insight into DED1 structure and interactions

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Characteristics of the earliest cross-neutralizing antibody response to HIV-1

Recent cross-sectional analyses of HIV-1+ plasmas have indicated that broadly cross-reactive neutralizing antibody responses are developed by 10%-30% of HIV-1+ subjects. The timing of the initial development of such anti-viral responses is unknown. It is also unknown whether the emergence of these responses coincides with the appearance of antibody specificities to a single or multiple regions of the viral envelope glycoprotein (Env). Here we analyzed the cross-neutralizing antibody responses in longitudinal plasmas collected soon after and up to seven years after HIV-1 infection. We find that anti-HIV-1 cross-neutralizing antibody responses first become evident on average at 2.5 years and, in rare cases, as early as 1 year following infection. If cross-neutralizing antibody responses do not develop during the first 2-3 years of infection, they most likely will not do so subsequently. Our results indicate a potential link between the development of cross-neutralizing antibody responses and specific activation markers on T cells, and with plasma viremia levels. The earliest cross-neutralizing antibody response targets a limited number of Env regions, primarily the CD4-binding site and epitopes that are not present on monomeric Env, but on the virion-associated trimeric Env form. In contrast, the neutralizing activities of plasmas from subjects that did not develop cross-neutralizing antibody responses target epitopes on monomeric gp120 other than the CD4-BS. Our study provides information that is not only relevant to better understanding the interaction of the human immune system with HIV but may guide the development of effective immunization protocols. Since antibodies to complex epitopes that are present on the virion-associated envelope spike appear to be key components of earliest cross-neutralizing activities of HIV-1+ plasmas, then emphasis should be made to elicit similar antibodies by vaccination.     

Expression of an exogenous peptide epitope genetically engineered in the variable domain of an immunoglobulin: implications for antibody and peptide folding.

Immunoglobulins bind antigens and express individual antigenic specificities mainly through residues located in hypervariable loops of their N-terminal domains. Hypervariable loops are kept in place by a molecular scaffold organized in a sandwich-like structure with two beta-sheets stabilized by a disulfide bridge (the immunoglobulin fold). This structural feature, together with the possibility of obtaining high level expression, extracellular secretion, easy purification and stability of the protein product, render immunoglobulin an ideal 'molecular vehicle' for the expression of exogenous peptides. Here we report on the engineering of an immunoglobulin expressing an exogenous epitope, the repetitive tetrapeptide Asn-Ala-Asn-Pro (NANP)3. By recombinant DNA techniques, we inserted three copies of the tetrapeptide (NANP)3 in the third hypervariable loop (D region) of an immunoglobulin heavy chain variable domain. We show that the engineered antibody was properly assembled and secreted. A panel of polyclonal and monoclonal antibodies, including anti-synthetic peptides and anti-(NANP)n antibodies, were used to study the molecular configuration of the engineered domain's surface. The results indicate that (i) the exogenous sequence did not appreciably alter the overall fold of the variable domain; and (ii) the inserted epitope folded with a configuration immunologically similar to the one assumed in the native protein, suggesting that short- and medium- rather than long-range interactions stabilized the structure of the (NANP)3 peptide in the folded protein. We propose this system for the expression of peptidic sequences, and their structural and functional analysis.     

Protein kinase C does not phosphorylate the externalized form of the transferrin receptor.

We have investigated the phosphorylation of transferrin receptors both in intact sheep reticulocytes and in isolated plasma membranes. Phosphorylation of the receptor in intact cells or isolated plasma membranes is stimulated by phorbol diesters, suggesting that protein kinase C may be involved. Identical [32P] phosphopeptide tryptic maps are formed in the presence and absence of phorbol diesters. Using heat-treated membranes (which are devoid of endogenous kinase activity) exogenous protein kinase C phosphorylates the same peptides as the endogenous kinase(s). During maturation of reticulocytes to erythrocytes, the transferrin receptor is released to the medium in vesicular form. In cells labelled with [32P]Pi, the released receptor is not labelled with 32P and the exocytosed vesicles do not phosphorylate receptor with [gamma-32P]ATP. The absence of 32P in the released receptor appears to be due to a change in the receptor, since, even in the presence of exogenous protein kinase C, the exocytosed receptor is phosphorylated to approximately 8% of the level obtained with receptors from the plasma membrane. These data suggest that during maturation and externalization the receptor is altered so that it loses its capacity to act as a substrate for exogenous protein kinase C as well as the endogenous kinase(s). This change may be a signal which segregates the receptor for externalization from the receptor pool remaining for transferrin recycling during the final stages of red cell maturation.     

A tyrosine-sulfated peptide derived from the heavy-chain CDR3 region of an HIV-1-neutralizing antibody binds gp120 and inhibits HIV-1 infection

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 entry. Human antibodies that recognize the CCR5-binding region of gp120 are also modified by tyrosine sulfation, which is necessary for their ability to neutralize HIV-1. Here we demonstrate that a sulfated peptide derived from the CDR3 region of one of these antibodies, E51, can efficiently bind gp120. Association of this peptide, pE51, with gp120 requires tyrosine sulfation and is enhanced by, but not dependent on, CD4. Alteration of any of four pE51 tyrosines, or alteration of gp120 residues 420, 421, or 422, critical for association with CCR5, prevents gp120 association with pE51. pE51 neutralizes HIV-1 more effectively than peptides based on the CCR5 amino terminus and may be useful as a fusion partner with other protein inhibitors of HIV-1 entry. Our data provide further insight into the association of the CCR5 amino terminus with gp120, show that a conserved, sulfate-binding region of gp120 is accessible to inhibitors in the absence of CD4, and suggest that soluble mimetics of CCR5 can be more effective than previously appreciated.     

Carbohydrate side chains of Rauscher leukemia virus envelope glycoproteins are not required to elicit a neutralizing antibody response.

Antisera raised against Rauscher leukemia virus (R-MuLV) contain a preponderance of antibodies against glycoprotein gp70 that are dependent on the presence of carbohydrate side chains for reactivity, as judged by immunoprecipitation or Western blotting. However, the majority of neutralizing antibodies were not dependent on the presence of carbohydrate, as indicated by (i) the ability of deglycosylated R-MuLV to adsorb neutralizing antibody from sera as efficiently as glycosylated R-MuLV and (ii) the ability of deglycosylated R-MuLV to induce neutralizing antibody responses when injected into rabbits. Moreover, a faster response was obtained with deglycosylated R-MuLV than with untreated control virus in the latter experiments. The results indicate that the neutralizing antibodies are a discrete subpopulation of the total antibody response. Furthermore, the carbohydrate moieties appear to afford protection to the virion during infection, rather than serve as a target for neutralization.     

In vitro HIV-1 selective integration into the target sequence and decoy-effect of the modified sequence

Although there have been a few reports that the HIV-1 genome can be selectively integrated into the genomic DNA of cultured host cell, the biochemistry of integration selectivity has not been fully understood. We modified the in vitro integration reaction protocol and developed a reaction system with higher efficiency. We used a substrate repeat, 5'-(GTCCCTTCCCAGT)(n)(ACTGGGAAGGGAC)(n)-3', and a modified sequence DNA ligated into a circular plasmid. CAGT and ACTG (shown in italics in the above sequence) in the repeat units originated from the HIV-1 proviral genome ends. Following the incubation of the HIV-1 genome end cDNA and recombinant integrase for the formation of the pre-integration (PI) complex, substrate DNA was reacted with this complex. It was confirmed that the integration selectively occurred in the middle segment of the repeat sequence. In addition, integration frequency and selectivity were positively correlated with repeat number n. On the other hand, both frequency and selectivity decreased markedly when using sequences with deletion of CAGT in the middle position of the original target sequence. Moreover, on incubation with the deleted DNAs and original sequence, the integration efficiency and selectivity for the original target sequence were significantly reduced, which indicated interference effects by the deleted sequence DNAs. Efficiency and selectivity were also found to vary discontinuously with changes in manganese dichloride concentration in the reaction buffer, probably due to its influence on the secondary structure of substrate DNA. Finally, integrase was found to form oligomers on the binding site and substrate DNA formed a loop-like structure. In conclusion, there is a considerable selectivity in HIV-integration into the specified sequence; however, similar DNA sequences can interfere with the integration process, and it is therefore difficult for in vivo integration to occur selectively in the actual host genome DNA.     

Induction of cytotoxic T lymphocytes in mice against the principal neutralizing domain of HIV-1 by immunization with an engineered T-cytotoxic-T-helper synthetic peptide construct.

Peptide constructs were engineered by colinear synthesis of two short synthetic peptide determinants; a determinant recognized by T helper cells (TDh) and a determinant recognized by T cytotoxic cells (TDc). Three types of constructs were synthesized: TDc-TDh, TDh-TDc, and TDh-KK-TDc, where KK are two lysine residues. In vivo immunization with free construct induced cytolytic lymphocytes (CTL) only in the case of TDc-TDh. However, immunization with spleen cells to which these constructs had been internalized by hypertonic shock, induced CTL activity in all three cases. No CTL could be induced after immunization with free TDc in either protocol. These results indicate that cell internalization of the construct might be essential for CTL induction, and also, that "help" from the TDh seems to be required.     

Profiling the allosteric response of an engineered beta-galactosidase to its effector, anti-HIV antibody

Escherichia coli beta-galactosidase responds enzymatically to antiviral antibodies when a viral antigenic peptide, acting as receptor, is conveniently displayed in the vicinity of the active site. The allosteric response of a beta-galactosidase molecular sensor containing a B-cell epitope from HIV has been finely dissected upon binding of an effector monoclonal antibody, within a wide range of standard concentrations of both enzyme and substrate. The topography of the enzymatic activation reveals a wide set of conditions in which the enzymatic response renders a signal over threefold the background, that is suitable for analytical biosensing. Moreover, at discrete enzyme-substrate coordinates, the effector antibody promotes an enhanced activation factor up to fivefold. The insertion of the 37-mer viral peptide between beta-galactosidase residues 795 and 796 is observed as inducer of the structural flexibility required for molecular sensing, whose dynamics and efficiency are intimately associated with the concentrations of enzyme and substrate, the two partners in the signal transduction event.     

Structure of a high-affinity "mimotope" peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies

The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.     

Insights into the oligomeric structure of the HIV-1 Vpu protein

The HIV-1-encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with host proteins to support the virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. Here, we report on the Vpu oligomeric organization under membrane and aqueous conditions and provide insights into how the Vpu environment affects the oligomer formation. For these studies, we designed a maltose-binding protein (MBP)-Vpu chimera protein and produced it in E. coli in soluble form. We analyzed this protein using analytical size-exclusion chromatography (SEC), negative staining electron microscopy (nsEM), and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, we found that MBP-Vpu formed stable oligomers in solution, seemingly driven by Vpu transmembrane domain self-association. A coarse modeling of nsEM data as well as SEC and EPR data suggests that these oligomers most likely are pentamers, similar to what was reported regarding membrane-bound Vpu. We also noticed reduced MBP-Vpu oligomer stability upon reconstitution of the protein in β-DDM detergent and mixtures of lyso-PC/PG or DHPC/DHPG. In these cases, we observed greater oligomer heterogeneity, with MBP-Vpu oligomeric order generally lower than in solution; however, larger oligomers were also present. Notably, we found that in lyso-PC/PG, above a certain protein concentration, MBP-Vpu assembles into extended structures, which had not been reported for Vpu. Therefore, we captured various Vpu oligomeric forms, which can shed light on Vpu quaternary organization. Our findings could be useful in understanding Vpu organization and function in cellular membranes and could provide information regarding the biophysical properties of single-pass transmembrane proteins.     

Non-active site changes elicit broad-based cross-resistance of the HIV-1 protease to inhibitors.

Three high level, cross-resistant variants of the HIV-1 protease have been analyzed for their ability to bind four protease inhibitors approved by the Food and Drug Administration (saquinavir, ritonavir, indinavir, and nelfinavir) as AIDS therapeutics. The loss in binding energy (DeltaDeltaG(b)) going from the wild-type enzyme to mutant enzymes ranges from 2.5 to 4.4 kcal/mol, 40-65% of which is attributed to amino acid substitutions away from the active site of the protease and not in direct contact with the inhibitor. The data suggest that non-active site changes are collectively a major contributor toward engendering resistance against the protease inhibitor and cannot be ignored when considering cross-resistance issues of drugs against the HIV-1 protease.     

Stabilization of the fibrous structure of an alpha-helix-forming peptide by sequence reversal

The alpha3-peptide, which comprises three repeats of the sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala and forms an amphipathic alpha-helix, is unique among various alpha-helix-forming peptides in that it assembles into fibrous structures that can be observed by transmission electron microscopy. As part of our investigation of the structure-stability relationships of the alpha3-peptide, we synthesized the r3-peptide, whose amino acid sequence is the reverse of that of the alpha3-peptide, and we investigated the effects of sequence reversal on alpha-helix stability and the formation of fibrous structures. Unexpectedly, the r3-peptide formed a more-stable alpha-helix and longer fibers than did the alpha3-peptide. The stability of the r3-peptide helix decreased when the ionic strength of the buffer was increased and when the pH of the buffer was adjusted to 2 or 12. These results suggest that the r3-peptide underwent a "magnet-like" oligomerization and that an increase in the charge-distribution inequality may be the driving force for the formation of fibrous structures.     

Amino acids and peptides. LVII. Synthetic peptide with a sequence of ribonuclease from Sulfolobus solfataricus, SSR(1-62), does not function as an RNase

The 62 residue peptide, SSR(1-62), whose sequence corresponds to that of ribonuclease (RNase) from Sulfolobus solfataricus, and its related peptides, SSR(1-22) and SSR(10-62), were chemically synthesized and their RNase activity and DNA-binding activity were examined. The RNase activity assay using yeast RNA or tRNA(fMet) as substrate showed that the synthetic peptide SSR(1-62) did not hydrolyze yeast RNA or tRNA(fMet). These data were not consistent with previous reports that both the native peptide isolated from S. solfataricus [Fusi et al. (1993) Eur. J. Biochem. 211, 305-311] and the recombinant peptide expressed in Escherichia coli [Fusi et al. (1995) Gene 154, 99-103] were able to hydrolyze tRNA(fMet). However, the synthetic SSR(1-62) exhibited DNA-binding activity. In the presence of synthetic SSR(1-62), the cleavage of DNA (plasmid pUCRh2-4) by restriction endonuclease (EcoRI) was not observed, suggesting that synthetic SSR(1-62) bound to DNA protected DNA from its enzymatic digestion. Neither SSR(1-22) nor SSR(10-62) prevented DNA from being cleaved by a restriction enzyme. These findings strongly suggest the importance of not only the N-terminal region of SSR(1-62) but also the C-terminal region for DNA-binding. Circular dichroism spectroscopy of synthetic SSR(1-62) indicated a beta-sheet conformation, in contrast with synthetic SSR(1-22), which exhibited an unordered conformation.     

The RNA annealing mechanism of the HIV-1 Tat peptide: conversion of the RNA into an annealing-competent conformation

The annealing of nucleic acids to (partly) complementary RNA or DNA strands is involved in important cellular processes. A variety of proteins have been shown to accelerate RNA/RNA annealing but their mode of action is still mainly uncertain. In order to study the mechanism of protein-facilitated acceleration of annealing we selected a short peptide, HIV-1 Tat(44-61), which accelerates the reaction efficiently. The activity of the peptide is strongly regulated by mono- and divalent cations which hints at the importance of electrostatic interactions between RNA and peptide. Mutagenesis of the peptide illustrated the dominant role of positively charged amino acids in RNA annealing--both the overall charge of the molecule and a precise distribution of basic amino acids within the peptide are important. Additionally, we found that Tat(44-61) drives the RNA annealing reaction via entropic rather than enthalpic terms. One-dimensional-NMR data suggest that the peptide changes the population distribution of possible RNA structures to favor an annealing-prone RNA conformation, thereby increasing the fraction of colliding RNA molecules that successfully anneal.