A chemically defined synthetic vaccine model for HIV-1.

Multiple Ag peptide (MAP) system without the use of a protein carrier was used as a vaccine model in three species of animals. Synthetic peptides from the V3 region of the gp120 of IIIB, RF and MN HIV-1 isolates were used as the Ag. MAP consisting of various chain lengths, from 11 to 24 residues, were prepared in a monoepitope configuration containing four repeats of each individual peptide. In parallel, they were synthesized in a diepitope configuration adding at the carboxyl-terminus of the V3 peptides a conserved sequence, known to be a Th cell epitope of gp120. The antibody response elicited by the monoepitope constructs was species-dependent. Rabbits produced immunity against all nine peptides, whereas mice were strongly reactive mainly to the longest sequence of the IIIB isolate. The immune response of guinea pigs was intermediate to those of rabbits and mice. Diepitope MAPs were immunogenic in all three species and elicited significantly higher titers than those raised by the immunization with the monoepitope MAPs. The response was type specific; the high-titered antibodies were reactive mostly against the isolate from which the peptides were derived, with a small cross-reactivity in ELISA between IIIB and RF strains. The dominant antigenic site of the B cell epitope, IIIB sequence, was located at the amino and central part of the MAP and a sequence overlapping the putative V3 reverse-turn was particularly reactive with the raised antibodies. Moreover, sera from the immunized animals inhibited virus-dependent cell fusion. These results show that MAP, with a chemically defined structure and without the use of a protein carrier, can be potentially useful for the design of synthetic HIV-1 vaccine candidates.     

Characterization update of HIV-1 M subtypes diversity and proposal for subtypes A and D sub-subtypes reclassification

Background

The large and constantly evolving HIV-1 pandemic has led to an increasingly complex diversity. Because of some taxonomic difficulties among the most diverse HIV-1 subtypes, and taking advantage of the large amount of sequence data generated in the recent years, we investigated novel lineage patterns among the main HIV-1 subtypes.

Results

All HIV full-length genomes available in public databases were analysed (n?=?2017). Maximum likelihood phylogenies and pairwise genetic distance were obtained. Clustering patterns and mean distributions of genetic distances were compared within and across the current groups, subtypes and sub-subtypes of HIV-1 to detect and analyse any divergent lineages within previously defined HIV lineages. The level of genetic similarity observed between most HIV clades was deeply consistent with the current classification. However, both subtypes A and D showed evidence of further intra-subtype diversification not fully described by the nomenclature system at the time and could be divided into several distinct sub-subtypes.

Conclusions

With this work, we propose an updated nomenclature of sub-types A and D better reflecting their current genetic diversity and evolutionary patterns. Allowing a more accurate nomenclature and classification system is a necessary step for easier subtyping of HIV strains and a better detection or follow-up of viral epidemiology shifts.

     

A model of HIV-1 transmission for urban areas of Africa

A model of the transmission dynamics of HIV-1, appropriate to urban areas of Africa, is presented and its behaviour explored through numerical studies. The model is a two-sex model with age-dependent demographic and behavioural parameters. Adults are classified by age, sex, risk group, and epidemiologic status. HIV-1 is transmitted to adults heterosexually, and to infants and children vertically and parenterally. Numerical studies show that, while AIDS will slow population growth, growth rates do not become negative for reasonable parameter values. The sex and age patterns of infection are explored, as is the potential economic impact of changes in the sex and age composition of the population.     

Westerdykella globosa,a proposal for a new combination

The surface ornamentation of ascospores ofPreussia globosa was compared in an isolate from paddy soil in Japan and a culture derived from the holotype. The ascospores of two cultures were characterized by the surface ornamentation of a single, semicircular spiral ridge. This new finding strongly suggested that the fungus should be transferred to the genusWesterdykella. Therefore, the morphological and cultural characters of the fungus were re-examined, and the new combinationWesterdykella globosa is herein proposed.     

A new structural theme in C2-symmetric HIV-1 protease inhibitors: ortho-substituted P1/P1' side chains

In this report, the rapid syntheses of 24 novel C2-symmetric HIV-1 protease inhibitors are described. Two ortho-iodobenzyloxy containing C-terminal duplicated inhibitors served as starting materials for microwave-enhanced palladium(0)-catalyzed carbon-carbon bond forming reactions (Suzuki, Sonogashira, Heck, and Negishi). Highly potent inhibitors equipped with ortho-functionalized P1/P1' side chains as the structural theme were identified. Computational efforts were applied to study the binding mode of this class of inhibitors and to establish structure-activity relationships. The overall orientation of the inhibitors in the active site was reproduced by docking which suggested three possible conformations of the P1/P1' groups of which two seem more plausible.     

A structural model of the HIV-1 Rev-integrase complex: the molecular basis of integrase regulation by Rev

The HIV-1 Rev and integrase (IN) proteins control important functions in the viral life cycle. We have recently discovered that the interaction between these proteins results in inhibition of IN enzymatic activity. Peptides derived from the Rev and IN binding interfaces have a profound effect on IN catalytic activity: Peptides derived from Rev inhibit IN, while peptides derived from IN stimulate IN activity by inhibiting the Rev-IN interaction. This inhibition leads to multi integration, genomic instability and specific death of virus-infected cells. Here we used protein docking combined with refinement and energy function ranking to suggest a structural model for the Rev-IN complex. Our results indicate that a Rev monomer binds IN at two sites that match our experimental binding data: (1) IN residues 66-80 and 118-128; (2) IN residues 174-188. According to our model, IN binds Rev and its cellular cofactor, lens epithelium derived growth factor (LEDGF), through overlapping interfaces. This supports previous observations that IN is regulated by a tight interplay between Rev and LEDGF. Rev may bind either the IN dimer or tetramer. Accordingly, Rev is suggested to inhibit IN by two possible mechanisms: (i) shifting the oligomerization equilibrium of IN from an active dimer to an inactive tetramer; (ii) displacing LEDGF from IN, resulting in inhibition of IN binding to the viral DNA. Our model is expected to contribute to the development of lead compounds that inhibit the Rev-IN interaction and thus lead to multi-integration of viral cDNA and consequently to apoptosis of HIV-1 infected cells.     

A proposal for a goodness-of-fit test to the Arnason-Schwarz multisite capture-recapture model

In an analysis of capture-recapture data, the identification of a model that fits is a critical step. For the multisite (also called multistate) models used to analyze data gathered at several sites, no reliable test for assessing fit is currently available. We propose a test for the JMV model, a simple generalization of the Arnason-Schwarz (AS) model, in the form of interpretable contingency tables. For the AS model, we suggest complementing the test for the JMV model with a likelihood ratio test of AS vs. JMV. The examination of an example leads us to propose further a partitioning that emphasizes the role of the memory model of Brownie et al. (1993 Biometrics 49, 1173-1187) as a biologically more plausible alternative to the AS model.     

A proposed molecular model for the carboxy terminus of HIV-1 gp120 showing structural features consistent with the presence of a T-cell alloepitope.

A computer graphics molecular model of the C terminus of gp120 of HIV has been constructed using predicted secondary structure based on homologies with proteins for which X-ray crystallographic data have been published. The model shows sequences known to be important in CD4 binding in close proximity to regions with a high probability of forming alpha helical and beta strand motifs. The orientation adopted by these domains approximates to the known 3D structure of HLA-A2 alpha 2 chain without constraints based on HLA-A2 as a template being introduced. The model may therefore represent an energetically favourable conformation for a part of gp120 which mimics the binding domain for the T-cell receptor on MHC molecules. Recognition of gp120 as an alloepitope in high affinity association with CD4 would explain many of the sequelae of acquired immune deficiency on HIV infection.     

Fighting a virus with a virus: a dynamic model for HIV-1 therapy

A mathematical model examined a potential therapy for controlling viral infections using genetically modified viruses. The control of the infection is an indirect effect of the selective elimination by an engineered virus of infected cells that are the source of the pathogens. Therefore, this engineered virus could greatly compensate for a dysfunctional immune system compromised by AIDS. In vitro studies using engineered viruses have been shown to decrease the HIV-1 load about 1000-fold. However, the efficacy of this potential treatment for reducing the viral load in AIDS patients is unknown. The present model studied the interactions among the HIV-1 virus, its main host cell (activated CD4+ T cells), and a therapeutic engineered virus in an in vivo context; and it examined the conditions for controlling the pathogen. This model predicted a significant drop in the HIV-1 load, but the treatment does not eradicate HIV. A basic estimation using a currently engineered virus indicated an HIV-1 load reduction of 92% and a recovery of host cells to 17% of their normal level. Greater success (98% HIV reduction, 44% host cells recovery) is expected as more competent engineered viruses are designed. These results suggest that therapy using viruses could be an alternative to extend the survival of AIDS patients.     

Synthesis of a new class of HIV-1 inhibitors

A new family of molecules potentially inhibitors of the HIV-1 Tat-TAR complex was prepared. These compounds are constituted by dinucleotide analogs (PNA dimer) bound, through a linker, to an arginine residue. In this series, several molecules inhibit viral development in cell culture with a micromolar IC50 and without cellular toxicity until 200 microM concentration.     

A structural and thermodynamic escape mechanism from a drug resistant mutation of the HIV-1 protease

The efficacy of HIV-1 protease inhibition therapies is often compromised by the appearance of mutations in the protease molecule that lower the binding affinity of inhibitors while maintaining viable catalytic activity and substrate affinity. The V82F/I84V double mutation is located within the binding site cavity and affects all protease inhibitors in clinical use. KNI-764, a second-generation inhibitor currently under development, maintains significant potency against this mutation by entropically compensating for enthalpic losses, thus minimizing the loss in binding affinity. KNI-577 differs from KNI-764 by a single functional group critical to the inhibitor response to the protease mutation. This single difference changes the response of the two inhibitors to the mutation by one order of magnitude. Accordingly, a structural understanding of the inhibitor response will provide important guidelines for the design of inhibitors that are less susceptible to mutations conveying drug resistance. The structures of the two compounds bound to the wild type and V82F/I84V HIV-1 protease have been determined by X-ray crystallography at 2.0 A resolution. The presence of two asymmetric functional groups, linked by rotatable bonds to the inhibitor scaffold, allows KNI-764 to adapt to the mutated binding site cavity more readily than KNI-577, with a single asymmetric group. Both inhibitors lose about 2.5 kcal/mol in binding enthalpy when facing the drug-resistant mutant protease; however KNI-764 gains binding entropy while KNI-577 loses binding entropy. The gain in binding entropy by KNI-764 accounts for its low susceptibility to the drug-resistant mutation. The heat capacity change associated with binding becomes more negative when KNI-764 binds to the mutant protease, consistent with increased desolvation. With KNI-577, the opposite effect is observed. Structurally, the crystallographic B factors increase for KNI-764 when it is bound to the drug-resistant mutant. The opposite is observed for KNI-577. Consistent with these observations, it appears that KNI-764 is able to gain binding entropy by a two-fold mechanism: it gains solvation entropy by burying itself deeper within the binding pocket and gains conformational entropy by losing interaction with the protease.     

Structure of the HIV-1 5' untranslated region dimer alone and in complex with gold nanocolloids: support of a TAR-TAR-containing 5' dimer linkage site (DLS) and a 3' DIS-DIS-containing DLS

The formation of a genomic RNA dimer is critical for the HIV-1 replication cycle, and dimerization is known to initiate within the 5'UTR (5' untranslated region) of the viral RNA. However, the 5'UTR constitutes the 335 terminal nucleotides, and because of this considerable size, it has been difficult to study the global structure using conventional structural methods. Here, the atomic force microscope has been used to directly visualize the dimer formed from RNAs including HIV-1 nucleotides 1-744. Gold nanocolloids were deposited on the primer binding site regions in the dimer as an internal control. The dimer showed distinct ring morphology with up to two gold nanocolloids deposited within the ring and one or two strands extending from the ring. This morphology implies a dimer including a DIS-DIS (dimerization initiation site)-containing 3' dimer linkage site (DLS) and a TAR-TAR (trans-activation region)-containing 5'DLS. Furthermore, the dimer was formed under the influence of Mg(2+) and was imaged with an atomic force microscope under buffer conditions. The overall ring morphology containing a 5'DLS and a 3'DLS with one or two strands extending from it was conserved in these atomic force microscopy images. This indicates that the observed dimer morphology is physiologically significant. Moreover, evidence of multiple dimer interstrand contacts downstream of the major splice donor were observed, which indicates a component in the selection of full-length genomic RNA in dimer formation during virion packaging.     

A structural model for ovine rhodopsin

An experimentally testable structure for ovine rhodopsin has been modelled from a combination of several secondary-structure prediction methods. The proposed structure agrees well with available experimental data. The model envisages seven transmembrane segments that are largely, but not entirely, α-helical. The prediction of roughly adjacent regions of more irregular structure within these segments (which could introduce significant changes in helix pitch or rotation) may provide a good working model for considering the structural mobility of the protein.     

A structure-based approach to a synthetic vaccine for HIV-1

The generation of neutralizing antibodies by peptide immunization is dependent on achieving conformational compatibility between antibodies and native protein. Consequently, approaches are needed for developing conformational mimics of protein neutralization sites. We replace putative main-chain hydrogen bonds (NH --> O=CRNH) with a hydrazone link (N-N=CH-CH(2)CH(2)) and scan constrained peptides for fit with neutralizing monoclonal antibodies (MAbs). To explore this approach, a V3 MAb 58.2 that potently neutralizes T-cell lab-adapted HIV-1(MN) was used to identify a cyclic peptide, [JHIGPGR(Aib)F(D-Ala)GZ]G-NH(2) (loop 5), that binds with >1000-fold higher affinity than the unconstrained peptide. NMR structural studies suggested that loop 5 stabilized beta-turns at GPGR and R(Aib)F(D-Ala) in aqueous solvent implying considerable conformational mimicry of a Fab 58.2 bound V3 peptide determined by X-ray crystallography [Stanfield, R. L. et al. (1999) Structure 142, 131-142]. Rabbit polyclonal antibodies (PAbs) generated to loop 5 but not to the corresponding uncyclized peptide bound the HIV-1(MN) envelope glycoprotein, gp120. When individual rabbit antisera were scanned with linear and cyclic peptides, further animal-to-animal differences in antibody populations were characterized. Loop 5 PAbs that most closely mimicked MAb 58.2 neutralized HIV-1(MN) with similar potency. These results demonstrate the remarkable effect that conformation can have on peptide affinity and immunogenicity and identify an approach that can be used to achieve these results. The implications for synthetic vaccine and HIV-1 vaccine research are discussed.     

A mathematical model of cell-to-cell spread of HIV-1 that includes a time delay

 We consider a two-dimensional model of cell-to-cell spread of HIV-1 in tissue cultures, assuming that infection is spread directly from infected cells to healthy cells and neglecting the effects of free virus. The intracellular incubation period is modeled by a gamma distribution and the model is a system of two differential equations with distributed delay, which includes the differential equations model with a discrete delay and the ordinary differential equations model as special cases. We study the stability in all three types of models. It is shown that the ODE model is globally stable while both delay models exhibit Hopf bifurcations by using the (average) delay as a bifurcation parameter. The results indicate that, differing from the cell-to-free virus spread models, the cell-to-cell spread models can produce infective oscillations in typical tissue culture parameter regimes and the latently infected cells are instrumental in sustaining the infection. Our delayed cell-to-cell models may be applicable to study other types of viral infections such as human T-cell leukaemia virus type 1 (HTLV-1). Received: 18 November 2000 / Published online: 28 February 2003 RID="*" ID="*" Research was partially supported by the NSERC and MITACS of Canada and a start-up fund from the College of Arts and Sciences at the University of Miami. On leave from Dalhousie University, Halifax, Nova Scotia, Canada. Current address: Department of Mathematics, Clarke College, Dubuque, Iowa 52001, USA Key words or phrases: HIV-1 – Cell-to-cell spread – Time delay – Stability – Hopf bifurcation – Periodicity