Vaccination of rabbits with an alkylated toxoid rapidly elicits potent neutralizing antibodies against botulinum neurotoxin serotype B.

New Zealand White (NZW) rabbits were immunized with several different nontoxic botulinum neurotoxin serotype B (BoNT/B) preparations in an effort to optimize the production of a rapid and highly potent, effective neutralizing antibody response. The immunogens included a recombinant heavy chain (rHc) protein produced in Escherichia coli, a commercially available formaldehyde-inactivated toxoid, and an alkylated toxoid produced by urea-iodoacetamide inactivation of the purified active toxin. All three immunogens elicited an antibody response to BoNT/B, detected by enzyme-linked immunosorbent assay (ELISA) and by toxin neutralization assay, by the use of two distinct mouse toxin challenge models. The induction period and the ultimate potency of the observed immune response varied for each immunogen, and the ELISA titer was not reliably predictive of the potency of toxin neutralization. The kinetics of the BoNT/B-specific binding immune response were nearly identical for the formaldehyde toxoid and alkylated toxoid immunogens, but immunization with the alkylated toxoid generated an approximately 10-fold higher neutralization potency that endured throughout the study, and after just 49 days, each milliliter of serum was capable of neutralizing 10(7) 50% lethal doses of the toxin. Overall, the immunization of rabbits with alkylated BoNT/B toxoid appears to have induced a neutralizing immune response more rapid and more potent than the responses generated by vaccination with formaldehyde toxoid or rHc preparations.

Novel bimodular DNA aptamers with guanosine quadruplexes inhibit phylogenetically diverse HIV-1 reverse transcriptases.

DNA aptamers RT5, RT6 and RT47 form a group of related sequences that inhibit HIV-1 reverse transcriptase (RT). The essential inhibitory structure is identified here as bimodular, with a 5' stem-loop module physically connected to a 3'-guanosine quadruplex module. The stem-loop tolerates considerable sequence plasticity. Connections between the guanosine triplets in the quadruplex could be simplified to a single nucleotide or a nonnucleic acid linker, such as hexaethylene glycol. All 12 quadruplex guanosines are required in an aptamer retaining most of the original loop sequence from RT6; only 11 are required for aptamer R1T (single T residue in intra-quadruplex loops). Circular dichroism (CD) spectroscopy gave ellipticity minima and maxima at 240 nm and 264 nm, indicating a parallel arrangement of the quadruplex strands. The simplified aptamers displayed increased overall stability. An aptamer carrying the original intra-quadruplex loops from RT6 inhibited RT in K(+) buffers but not in Na(+) buffers and displayed significant CD spectral broadening in Na(+) buffers, while R1T inhibited RT in both buffers and displayed less broadening in Na(+) buffers. The bimodular ssDNA aptamers inhibited RT from diverse primate lentiviruses with low nM IC(50) values. These data provide insight into the requirements for broad-spectrum RT inhibition by nucleic acid aptamers.

Active site binding and sequence requirements for inhibition of HIV-1 reverse transcriptase by the RT1 family of single-stranded DNA aptamers.

Nucleic acid aptamers can potentially be developed as broad-spectrum antiviral agents. Single-stranded DNA (ssDNA) aptamer RT1t49 inhibits reverse transcriptases (RT) from HIV-1 and diverse lentiviral subtypes with low nanomolar values of Kd and IC50. To dissect the structural requirements for inhibition, RT-catalyzed DNA polymerization was measured in the presence of RT1t49 variants. Three structural domains were found to be essential for RT inhibition by RT1t49: a 5' stem (stem I), a connector and a 3' stem (stem II) capable of forming multiple secondary structures. Stem I tolerates considerable sequence plasticity, suggesting that it is recognized by RT more by structure than by sequence-specific contacts. Truncating five nucleotides from the 3' end prevents formation of the most stable stem II structure, yet has little effect on IC50 across diverse HIV-1, HIV-2 and SIV(CPZ) RT. When bound to wild-type RT or an RNase H active site mutant, site-specifically generated hydroxyl radicals cleave after nucleotide A32. Cleavage is eliminated by either of two polymerase (pol)-active site mutants, strongly suggesting that A32 lies within the RT pol-active site. These data suggest a model of ssDNA aptamer-RT interactions and provide an improved molecular understanding of a potent, broad-spectrum ssDNA aptamer.

Single-stranded DNA aptamer RT1t49 inhibits RT polymerase and RNase H functions of HIV type 1, HIV type 2, and SIVCPZ RTs.

Natural and selected resistance of HIV-1 to current anti-HIV drugs continues to pose serious problems to the development of HIV-1 antivirals. The viral reverse transcriptase (RT) is a proven therapeutic target. Single-stranded RNA and DNA (ssRNA and ssDNA) aptamers have been selected that specifically and potently inhibit RT function. In particular, the ssDNA aptamer RT1t49 was previously selected to recognize the RT from a subtype B strain of HIV-1 and binds with a reported K(d) of 4 nM. In the present work, we show that RT1t49 inhibits recombinant RT cloned from diverse branches of the primate lentiviral family. Aptamer concentrations required for half-maximal inhibition of all HIV-1, HIV-2, and SIV(CPZ) RTs assayed were in the low-to mid-nanomolar range for both polymerase and RNase H activities. Using pre-steady-state and order-of-addition kinetic analyses, we also established that this ssDNA aptamer competes with primer-template for access to RT, and that addition of a nucleoside analog RT inhibitor (NRTI) to the in vitro reaction enhanced the overall effectiveness of both drugs, while nonnucleoside analog RT inhibitors (NNRTIs) exhibited simple additivity. This is the first demonstration of universal inhibition of HIV and SIV(cpz) RTs by a nucleic acid aptamer and supports previous reports suggesting that resistance to RT1t49 may be exceptionally infrequent.

Cross-clade inhibition of recombinant human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus SIVcpz reverse transcriptases by RNA pseudoknot aptamers.

Reverse transcriptase (RT) remains a primary target in therapies directed at human immunodeficiency virus type 1 (HIV-1). RNA aptamers that bind RT from HIV-1 subtype B have been shown to protect human cells from infection and to reduce viral infectivity, but little is known about the sensitivity of the inhibition to amino sequence variations of the RT target. Therefore, we assembled a panel of 10 recombinant RTs from phylogenetically diverse lentiviral isolates (including strains of HIV-1, simian immunodeficiency virus SIVcpz, and HIV-2). After validating the panel by measuring enzymatic activities and inhibition by small-molecule drugs, dose-response curves for each enzyme were established for four pseudoknot RNA aptamers representing two structural subfamilies. All four aptamers potently inhibited RTs from multiple HIV-1 subtypes. For aptamers carrying family 1 pseudoknots, natural resistance was essentially all-or-none and correlated with the identity of the amino acid at position 277. In contrast, natural resistance to aptamers carrying the family 2 pseudoknots was much more heterogeneous, both in degree (gradation of 50% inhibitory concentrations) and in distribution across clades. Site-directed and subunit-specific mutagenesis identified a common R/K polymorphism within the p66 subunit as a primary determinant of resistance to family 1, but not family 2, pseudoknot aptamers. RNA structural diversity therefore translates into a nonoverlapping spectrum of mutations that confer resistance, likely due to differences in atomic-level contacts with RT.

Multiple-turnover thio-ATP hydrolase and phospho-enzyme intermediate formation activities catalyzed by an RNA enzyme.

Ribozymes that phosphorylate internal 2'-OH positions mimic the first mechanistic step of P-type ATPase enzymes by forming a phospho-enzyme intermediate. We previously described 2'-autophosphorylation and autothiophosphorylation by the 2PTmin3.2 ribozyme. In the present work we demonstrate that the thiophosphorylated form of this ribozyme can de-thiophosphorylate in the absence of ATPgammaS. Identical ionic conditions yield a thiophosphorylated strand when ATPgammaS is included, thus effecting a net ATPgammaS hydrolysis. The de-thiophosphorylation step is nearly independent of pH over the range of 6.3-8.5 and does not require a specifically folded RNA structure, but this step is greatly stimulated by transition metal ions. By monitoring thiophosphate release, we observe 29-46 ATPgammaS hydrolyzed per ribozyme strand in 24 h, corresponding to a turnover rate of 1.2-2.0 h(-1). The existence of an ATP- (or thio-ATP-)powered catalytic cycle raises the possibility of using ribozymes to transduce chemical energy into mechanical work for nucleic acid nanodevices.

Differential susceptibility of HIV-1 reverse transcriptase to inhibition by RNA aptamers in enzymatic reactions monitoring specific steps during genome replication.

Nucleic acid aptamers to HIV-1 reverse transcriptase (RT) are potent inhibitors of DNA polymerase function in vitro, and they have been shown to inhibit viral replication when expressed in cultured T-lymphoid lines. We monitored RT inhibition by five RNA pseudoknot RNA aptamers in a series of biochemical assays designed to mimic discrete steps of viral reverse transcription. Our results demonstrate potent aptamer inhibition (IC50 values in the low nanomolar range) of all RT functions assayed, including RNA- and DNA-primed DNA polymerization, strand displacement synthesis, and polymerase-independent RNase H activity. Additionally, we observe differences in the time dependence of aptamer inhibition. Polymerase-independent RNase H activity is the most resistant to long term aptamer suppression, and RNA-dependent DNA polymerization is the most susceptible. Finally, when DNA polymerization was monitored in the presence of an RNA aptamer in combination with each of four different small molecule inhibitors, significant synergy was observed between the aptamer and the two nucleoside analog RT inhibitors (azidothymidine triphosphate or ddCTP), whereas two non-nucleoside analog RT inhibitors showed either weak synergy (efavirenz) or antagonism (nevirapine). Together, these results support a model wherein aptamers suppress viral replication by cumulative inhibition of RT at every stage of genome replication.

HIV-1 inactivation by nucleic acid aptamers.

Although developments in small-molecule therapeutics for HIV-1 have been dramatic in recent years, the rapid selection of drug-resistant viral strains and the adverse side effects associated with long-term exposure to current treatments propel continued exploration of alternative anti-HIV-1 agents. Non-coding nucleic acids have emerged as potent inhibitors that dramatically suppress viral function both in vitro and in cell culture. In particular, RNA and DNA aptamers inhibit HIV-1 function by directly interfering with essential proteins at critical stages in the viral replication cycle (Figure 1). Their antiviral efficacy is expected to be a function, in part, of the biochemical properties of the aptamer-target interaction. Accordingly, we present an overview of biochemical and cell culture analyses of the expanding list of aptamers targeting HIV-1. Our discussion focuses on the inhibition of viral enzymes (reverse transcription, proteolytic processing, and chromosomal integration), viral expression (Rev/RRE and Tat/TAR), viral packaging (p55Gag, matrix and nucleocapsid), and viral entry (gp120) (Table 1). Additional nucleic acid-based strategies for inactivation of HIV-1 function (including RNAi, antisense, and ribozymes) have also demonstrated their utility. These approaches are reviewed in other chapters of this volume and elsewhere.

Evolutionary landscapes for the acquisition of new ligand recognition by RNA aptamers.

The evolution of ligand specificity underlies many important problems in biology, from the appearance of drug resistant pathogens to the re-engineering of substrate specificity in enzymes. In studying biomolecules, however, the contributions of macromolecular sequence to binding specificity can be obscured by other selection pressures critical to bioactivity. Evolution of ligand specificity in vitro--unconstrained by confounding biological factors--is addressed here using variants of three flavin-binding RNA aptamers. Mutagenized pools based on the three aptamers were combined and allowed to compete during in vitro selection for GMP-binding activity. The sequences of the resulting selection isolates were diverse, even though most were derived from the same flavin-binding parent. Individual GMP aptamers differed from the parental flavin aptamers by 7 to 26 mutations (20 to 57% overall change). Acquisition of GMP recognition coincided with the loss of FAD (flavin-adenine dinucleotide) recognition in all isolates, despite the absence of a counter-selection to remove FAD-binding RNAs. To examine more precisely the proximity of these two activities within a defined sequence space, the complete set of all intermediate sequences between an FAD-binding aptamer and a GMP-binding aptamer were synthesized and assayed for activity. For this set of sequences, we observe a portion of a neutral network for FAD-binding function separated from GMP-binding function by a distance of three mutations. Furthermore, enzymatic probing of these aptamers revealed gross structural remodeling of the RNA coincident with the switch in ligand recognition. The capacity for neutral drift along an FAD-binding network in such close approach to RNAs with GMP-binding activity illustrates the degree of phenotypic buffering available to a set of closely related RNA sequences--defined as the set's functional tolerance for point mutations--and supports neutral evolutionary theory by demonstrating the facility with which a new phenotype becomes accessible as that buffering threshold is crossed.