Biomedical subjects

J L Spouge

Publications and source records attributed to J L Spouge.

14 recordsLinked to original sources

PubMed1992

Statistical analysis of sparse infection data and its implications for retroviral treatment trials in primates.

Reports on retroviral primate trials rarely publish any statistical analysis. Present statistical methodology lacks appropriate tests for these trials and effectively discourages quantitative assessment. This paper describes the theory behind VACMAN, a user-friendly computer program that calculates statistics for in vitro and in vivo infectivity data. VACMAN's analysis applies to many retroviral trials using i.v. challenges and is valid whenever the viral dose-response curve has a particular shape. Statistics from actual i.v. retroviral trials illustrate some unappreciated principles of effective animal use: dilutions other than 1:10 can improve titration accuracy; infecting titration animals at the lowest doses possible can lower challenge doses; and finally, challenging test animals in small trials with more virus than controls safeguards against false successes, "reuses" animals, and strengthens experimental conclusions. The theory presented also explains the important concept of viral saturation, a phenomenon that may cause in vitro and in vivo titrations to agree for some retroviral strains and disagree for others.

Animals↗

PubMed1992

Factors underlying spontaneous inactivation and susceptibility to neutralization of human immunodeficiency virus.

To determine the factors governing inactivation and neutralization, physical, chemical, and biological assays were performed on a molecular clone of human immunodeficiency type 1 (HIV-1HXB3). This included quantitative electron microscopy, gp120 and p24 enzyme-linked immunosorbent assays, reverse, transcriptase assays, and quantitative infectivity assays. For freshly harvested stocks, the ratio of infectious to noninfectious viral particles ranged from 10(-4) to 10(-7) in viral stocks containing 10(9) to 10(10) physical particles per milliliter. There were relatively few gp120 knobs per HIV particle, mean approximately 10 when averaged over the total particle count. Each HIV particle contained a mean approximately 5 x 10(-17) g of p24 and approximately 2 x 10(-16) g of RNA polymerase, corresponding to about 1200 and 80 molecules, respectively. The spontaneous shedding of gp120 envelope proteins from virions was exponential, with a half-life approximately 30 hr. The loss of RNA polymerase activity in virons was also exponential, with a half-life approximately 40 hr. The physical breakup of virions and the dissolution of p24 core proteins were slow (half-life greater than 100 hr) compared to the gp120 shedding and polymerase loss rates. The decay of HIV-1 infectivity was found to obey superimposed single- and multihit kinetics. At short preincubation times, the loss of infectivity correlated with spontaneous shedding of gp120 from virions. At longer times, an accelerating decay rate indicated that HIV requires a minimal number of gp120 molecules for efficient infection of CD4+ cells. The blocking activity of recombinant soluble CD4 (sCD4) and phosphonoformate (foscarnet) varied with the number of gp120 molecules and number of active RNA polymerase molecules per virion, respectively. These results demonstrate that the physical state of virions greatly influences infectivity and neutralization. The knowledge gained from these findings will improve the reliability of in vitro assays, enhance the study of wild-type strains, and facilitate the evaluation of potential HIV therapeutics and vaccines.

CD4 Antigens↗

PubMed1991

Fast optimal alignment.

Algorithms often align sequences by minimizing a cost. Such algorithms usually operate by aligning successively longer subsequences until they finish the alignment. Efficient algorithms, such as those of Fickett and Ukkonen, speed the computation by ignoring bad subalignments. A general principle underlies the efficiency of these two algorithms: inequalities can direct computations to promising subalignments. Hence inequalities can be used to suggest alignment algorithms. Inequalities for unweighted end-gaps, affine and concave gap weights, etc., are discussed, and empirical results evaluating new algorithms for single indel costs and weighted end-gaps are presented. Empirical results show the new algorithms are, under certain circumstances, much faster than known algorithms.

Algorithms↗

PubMed1991

Blocking of human immunodeficiency virus infection depends on cell density and viral stock age.

Quantitative infectivity assays were used to study how the blocking activity of soluble CD4 (sCD4) is affected by sCD4 concentration, target cell density, and viral stock age. During incubation with 20 nM sCD4, human immunodeficiency virus type 1 (HIV-1) stocks underwent irreversible inactivation. In contrast, inactivation with 2 nM sCD4 was almost entirely reversible. At lower sCD4 concentrations (less than or equal to 2 nM) and target cell densities of 6.25 x 10(4) ml-1, sCD4 blocking activity for HIV-1 gave a gp120-sCD4 association constant (Kassoc) of 1.7 x 10(9) M-1, which agrees with chemical measurements. At the higher density of 1.6 x 10(7) cells ml-1, however, the blocking activity was 20-fold less. During incubation of HIV-1 stock optimized for infectivity by rapid harvest, sCD4 blocking activity increased 20-fold during a 3-h window. These results show that competitive blocking activity depends strongly on target cell density and virion age. Thus, unappreciated variations in HIV stocks and assay conditions may hinder comparisons of blockers from laboratory to laboratory, and the age of HIV challenge stocks may influence studies of drug and vaccine efficacy. The results also suggest that blocking of viral particles in lymphoid compartments will require very high competitive blocker concentrations, which may explain the refractory outcomes from sCD4-based drug trials in humans.

Acquired Immunodeficiency Syndrome↗

PubMed1990

HIV requires multiple gp120 molecules for CD4-mediated infection.

Binding of glycoprotein gp120 to the T cell-surface receptor CD4 is a crucial step in CD4-dependent infection of a target cell by the human immunodeficiency virus (HIV). Blocking some or all gp120 molecules on the viral surface should therefore inhibit infection. Consequently, competitive receptor inhibitors, such as soluble synthetic CD4 (sCD4), synthetic CD4 peptides and immunoglobulins, have been investigated in vitro and in vivo, but little is known about the molecular mechanisms of these inhibitors. We have now quantitatively examined blocking by soluble CD4 in the hope of gaining insight into the complex process of viral binding, adsorption and penetration. At low sCD4 concentrations, the inhibition in three HIV strains is proportional to the binding of gp120. The biological association constant (gp120-sCD4 Kassoc) for HIV-2NIHZ is (8.5 +/- 0.5) x 10(7) M-1, whereas Kassoc for HIV-1HXB3 (1.4 +/- 0.2) and HIV-1MN (1.7 +/- 0.1) x 10(9) M-1 are 15-20-fold larger. For all three viral strains, the biological Kassoc from infectivity assays is comparable to the chemical Kassoc. The inhibitory action of sCD4 at high concentrations, however, is not fully explained by simple proportionality with the binding to gp120. Positive synergy in blocking of infection occurs after about half the viral gp120s molecules are occupied, and is identical for all three viral strains, despite the large differences in Kassoc. Our method of measuring the viral-cell receptor Kassoc directly from infectivity assays is applicable to immunoglobulins, to other viruses and to assays using primary or transformed cell lines.

CD4 Antigens↗

PubMed1989

Analytic results for quantifying HIV infectivity.

In a separate paper, we developed a mathematical model describing HIV infection and used it to suggest experiments for quantifying characteristic viral parameters. In this paper we generalize the model to any well-mixed assay system. We also present complete and rigorous derivations of fundamental results needed for the design and analysis of HIV infectivity assays. The model is applicable to infectious agents with multiple receptors for their target cell (e.g. HIV, Epstein-Barr virus and Plasmodium), and to blockers (both reversible and irreversible), as long as blocker and target cells are the same diffusion compartment.

Acquired Immunodeficiency Syndrome↗

PubMed1989

Quantifying the infectivity of human immunodeficiency virus.

We have developed a mathematical model that quantifies lymphocyte infection by human immunodeficiency virus (HIV) and lymphocyte protection by blocking agents such as soluble CD4. We use this model to suggest standardized parameters for quantifying viral infectivity and to suggest techniques for calculating these parameters from well-mixed infectivity assays. We discuss the implications of the model for our understanding of the infectious process and virulence of HIV in vivo.

Acquired Immunodeficiency Syndrome↗

PubMed1989

T cell multideterminant regions in the human immunodeficiency virus envelope: toward overcoming the problem of major histocompatibility complex restriction.

Helper T cell determinants should be an important component of an anti-human immunodeficiency virus (HIV) vaccine aimed at either antibody or cytotoxic T cell immunity. However, model protein studies have raised concern about the usefulness of any single determinant, because a given determinant is likely to be seen by only a small subset of major histocompatibility complex (MHC) types within the population. Here, we use 44 peptides, including ones predicted and not predicted on the basis of amphipathicity to be potential T cell sites, to locate T cell antigenic determinants recognized by mice of four MHC haplotypes immunized with the whole gp 160 envelope protein. Although the preselection of peptides necessitates caution in a statistical analysis, alpha-amphipathic peptides predominated among sites eliciting the strongest response. Although we have not tested the entire sequence, we have identified six multideterminant regions, in which overlapping peptides are recognized by mice of either three or all four MHC types. Four of the six regions have sequences relatively conserved among HIV-1 isolates. The existence of such multideterminant regions recognized by multiple MHC haplotypes suggests the possibility that use of peptides longer than a minimal determinant and containing several overlapping determinants may be a possible approach to circumvent the serious problem of MHC restriction in peptide vaccines aimed at eliciting T cell immunity.

Amino Acid Sequence↗

PubMed1987

Hydrophobicity scales and computational techniques for detecting amphipathic structures in proteins.

Protein segments that form amphipathic alpha-helices in their native state have periodic variation in the hydrophobicity values of the residues along the segment, with a 3.6 residue per cycle period characteristic of the alpha-helix. The assignment of hydrophobicity values to amino acids (hydrophobicity scale) affects the display of periodicity. Thirty-eight published hydrophobicity scales are compared for their ability to identify the characteristic period of alpha-helices, and an optimum scale for this purpose is computed using a new eigenvector method. Two of the published scales are also characterized by eigenvectors. We compare the usual method for detecting periodicity based on the discrete Fourier transform with a method based on a least-squares fit of a harmonic sequence to a sequence of hydrophobicity values. The two become equivalent for very long sequences, but, for shorter sequences with lengths commonly found in alpha-helices, the least-squares procedure gives a more reliable estimate of the period. The analog to the usual Fourier transform power spectrum is the "least-squares power spectrum", the sum of squares accounted for in fitting a sinusoid of given frequency to a sequence of hydrophobicity values. The sum of the spectra of the alpha-helices in our data base peaks at 97.5 degrees, and approximately 50% of the helices can account for this peak. Thus, approximately 50% of the alpha-helices appear to be amphipathic, and, of those that are, the dominant frequency at 97.5 degrees rather than 100 degrees indicates that the helix is slightly more open than previously thought, with the number of residues per turn closer to 3.7 than 3.6. The extra openness is examined in crystallographic data, and is shown to be associated with the C terminus of the helix. The alpha amphipathic index, the key quantity in our analysis, measures the fraction of the total spectral area that is under the 97.5 degrees peak, and is a characteristic of hydrophobicity scales that is consistent for different sets of helices. Our optimized scale maximizes the amphipathic index and has a correlation of 0.85 or higher with nine previously published scales. The most surprising feature of the optimized scale is that arginine tends to behave as if it were hydrophobic; i.e. in the crystallographic data base it has a tendency to be on the hydrophobic face of teh amphipathic helix. Although the scale is optimal only for predicting alpha-amphipathicity, it also ranks high in identifying beta-amphipathicity and in distinguishing interior from exterior residues in a protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Algorithms↗

PubMed1987

Prediction of immunodominant helper T cell antigenic sites from the primary sequence.

We have used a data base of 23 known immunodominant helper T cell antigenic sites located on 12 proteins to systematically develop an optimized algorithm for predicting T cell antigenic sites. The algorithm is based on the amphipathic helix model in which antigenic sites are postulated to be helices with one face predominantly polar and the opposite face predominantly apolar. Such amphipathic structures can form when the polarity of residues along the sequence varies with a more or less regular period. Hence they can be identified by methods (so called power spectrum procedures) that detect periodic variations in properties of a sequence. The choice of power spectrum procedure, hydrophobicity scale, and model parameters are examined. An algorithm is tested by comparing the predicted amphipathic segments with the locations of the known T cell sites, counting the number of matches, and calculating the probability of getting this number by chance alone. The optimum algorithm, which predicts the largest number of sites with the lowest chance probability, uses the Fauchere-Pliska hydrophobicity scale and a least squares fit of a sinusoid as its power spectrum procedure. By applying this algorithm, 18 of the 23 known sites are identified (75% sensitivity) with a high degree of significance (p less than 0.001). The success of the algorithm supports the hypothesis that stable amphipathic helices are fundamentally important in determining immunodominance. This approach may be of practical value in designing synthetic vaccines aimed at T cell immunity.

Algorithms↗

PubMed1987

Strong conformational propensities enhance T cell antigenicity.

The ability to predict T cell antigenic peptides would have important implications for the development of artificial vaccines. As a first step towards prediction, this report uses a new statistical technique to discover and evaluate peptide properties correlating with T cell antigenicity. This technique employs Monte Carlo computer experiments and is applicable to many problems involving protein or DNA. The technique is used to evaluate the contribution of various peptide properties to helper T cell antigenicity. The properties investigated include amphipathicities (alpha and beta), conformational propensities (alpha, beta, turn and coil), and the correlates of alpha-helices, such as the absence of helix-breakers and the positioning of the residues which stabilize alpha-helical dipoles. We also investigate segmental amphipathicity. (A peptide has this property when it contains at least two disjoint subpeptides, one hydrophobic, one hydrophilic.) Statistical correlations and stratifications assessed independent contributions to T cell antigenicity. The findings presented here have important implications for the manufacture of peptide vaccines. These implications are as follows: if possible, peptide vaccines should probably be those protein segments which have a propensity to form amphipathic alpha-helices, which do not have regions with a propensity to coil conformations, and which have a lysine at their COOH-terminus. The last two observations are of particular use in manufacturing peptides vaccines: they indicate where the synthetic peptides should be terminated. These implications are supported by the findings given below. The significances (p values) support the following statistical generalites about antigenic conformations: most helper T cell antigenic sites are amphipathic alpha-helices; alpha-helical amphipathicity and propensity to an alpha-helical conformation contribute independently to T cell antigenicity; there is evidence that some T cell antigenic sites are beta conformations instead of alpha-helices; T cell antigenic sites avoid random coiled conformations; and T cell antigenic sites are usually not segmentally amphipathic. alpha-Helical amphipathicity was significant, but segmental amphipathicity was not. This has implications for the dimensions of the structure interacting with the hydrophobic portion of an amphipathic T cell antigenic site. Lysines are unusually frequent at the COOH-terminal of T cell antigenic sites, even after accounting for tryptic digests. These lysines can stabilize alpha-helical peptides by a favorable interaction with alpha-helical dipoles.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Protein antigenic structures recognized by T cells: potential applications to vaccine design.

In summary, our results using the model protein antigen myoglobin indicated, in concordance with others, that helper T lymphocytes recognize a limited number of immunodominant antigenic sites of any given protein. Such immunodominant sites are the focus of a polyclonal response of a number of different T cells specific for distinct but overlapping epitopes. Therefore, the immunodominance does not depend on the fine specificity of any given clone of T cells, but rather on other factors, either intrinsic or extrinsic to the structure of the antigen. A major extrinsic factor is the MHC of the responding individual, probably due to a requirement for the immunodominant peptides to bind to the MHC of presenting cells in that individual. In looking for intrinsic factors, we noted that both immunodominant sites of myoglobin were amphipathic helices, i.e., helices having hydrophilic and hydrophobic residues on opposite sides. Studies with synthetic peptides indicated that residues on the hydrophilic side were necessary for T-cell recognition. However, unfolding of the native protein was shown to be the apparent goal of processing of antigen, presumably to expose something not already exposed on the native molecule, such as the hydrophobic sides of these helices. We propose that such exposure is necessary to interact with something on the presenting cell, such as MHC or membrane, where we have demonstrated the presence of antigenic peptides by blocking of presentation of biotinylated peptide with avidin. The membrane may serve as a short-term memory of peptides from antigens encountered by the presenting cell, for dynamic sampling by MHC molecules to be available for presentation to T cells. These ideas, together with the knowledge that T-cell recognition required only short peptides and therefore had to be based only on primary or secondary structure, not tertiary folding of the native protein, led us to propose that T-cell immunodominant epitopes may tend to be amphipathic structures. An algorithm to search for potential amphipathic helices from sequence information identified 18 of 23 known immunodominant T-cell epitopes from 12 proteins (p less than 0.001). Another statistical approach confirmed the importance of amphipathicity and also supported the importance of helical structure that had been proposed by others. It suggested that peptides able to form a stable secondary structure, especially a helix, more commonly formed immunodominant epitopes. We used this approach to predict potential immunodominant epitopes for induction of T-cell immunity in proteins of clinical relevance, such as the malarial circumsporozoite protein and the AIDS viral envelope.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens↗

PubMed1985

Improving sequence-matching algorithms by working from both ends.

Recent algorithms (e.g. Ukkonen, Fickett) align nucleic acid sequences (starting from the left) by bounding the allowed distance between subsequences by d, aligning, then incrementing d until all of both sequences are aligned. Aligning from both ends is more efficient. If the single-ended algorithm has computational cost CNk (C, k = constants; N = sequence length), the double-ended algorithm often has cost C(N/2)k.

Base Sequence↗

PubMed1985

On a model for the structure of circular mitochondrial genomes in higher plants.

The mitochondrial genome of some higher plants consists of a discrete set of circular molecules. The heterogeneity found in the number of circles and their size is puzzling. In some plants evidence exists that indicates that these circular DNA molecules undergo intermolecular and intramolecular recombination events. The result of these events is that two small circles can combine to give one large circle or one large circle can break up into two small circles. Here we pursue the idea that such recombination events are responsible for the variability in genome composition. Treating the recombination events as chemical reactions we derive the equilibrium size distribution of circles. In Brassica campestris there are two basic subunits, circles A and B, containing 135 and 83 kilobases, respectively. By restriction-enzyme mapping one can determine in a multimeric circle, the number of interfaces between A- and B-derived DNA. Using a combinatorial argument we predict the frequency of such interfaces and compare our predictions with published data. A number of suggestions are made for additional experimental tests of the recombinational theory of genomic diversity.

DNA, Circular↗