PAQ: Partition Analysis of Quasispecies.

MOTIVATION: The complexities of genetic data may not be accurately described by any single analytical tool. Phylogenetic analysis is often used to study the genetic relationship among different sequences. Evolutionary models and assumptions are invoked to reconstruct trees that describe the phylogenetic relationship among sequences. Genetic databases are rapidly accumulating large amounts of sequences. Newly acquired sequences, which have not yet been characterized, may require preliminary genetic exploration in order to build models describing the evolutionary relationship among sequences. There are clustering techniques that rely less on models of evolution, and thus may provide nice exploratory tools for identifying genetic similarities. Some of the more commonly used clustering methods perform better when data can be grouped into mutually exclusive groups. Genetic data from viral quasispecies, which consist of closely related variants that differ by small changes, however, may best be partitioned by overlapping groups. RESULTS: We have developed an intuitive exploratory program, Partition Analysis of Quasispecies (PAQ), which utilizes a non-hierarchical technique to partition sequences that are genetically similar. PAQ was used to analyze a data set of human immunodeficiency virus type 1 (HIV-1) envelope sequences isolated from different regions of the brain and another data set consisting of the equine infectious anemia virus (EIAV) regulatory gene rev. Analysis of the HIV-1 data set by PAQ was consistent with phylogenetic analysis of the same data, and the EIAV rev variants were partitioned into two overlapping groups. PAQ provides an additional tool which can be used to glean information from genetic data and can be used in conjunction with other tools to study genetic similarities and genetic evolution of viral quasispecies.

The organization of human leucocyte antigen class I epitopes in HIV genome products: implications for HIV evolution and vaccine design.

Knowledge of human leucocyte antigen (HLA) peptide binding motifs permits rapid selection of candidate viral protein fragments for induction of T cell-mediated immunity. A search for HLA class I peptide binding motifs in structural proteins of human immunodeficiency virus (HIV) of different genetic lineages provides a map of the genetic organization of potential T cell antigenic sites, and at the same time identifies all motifs in highly conserved regions of HIV-1 env, gag and pol. The density of motifs is anomalous at both the high and low end of the spectrum: local organization is characterized by clustering in relatively short regions, while large scale organization is characterized by anomalously long runs between motifs. The former is expected simply due to the fact that motifs often have overlapping anchor residue sets. A detailed statistical analysis of the latter, however, shows that the length of the runs cannot be accounted for by chance alone. Although motif clusters show no preference to be in either conserved or variable regions, low motif density stretches occur preferentially in variable portions of the protein sequence, which suggests that the virus may be mutating to evade the cellular arm of the immune system.

Consistency in structural energetics of protein folding and peptide recognition.

We report a new free energy decomposition that includes structure-derived atomic contact energies for the desolvation component, and show that it applies equally well to the analysis of single-domain protein folding and to the binding of flexible peptides to proteins. Specifically, we selected the 17 single-domain proteins for which the three-dimensional structures and thermodynamic unfolding free energies are available. By calculating all terms except the backbone conformational entropy change and comparing the result to the experimentally measured free energy, we estimated that the mean entropy gain by the backbone chain upon unfolding (delta Sbb) is 5.3 cal/K per mole of residue, and that the average backbone entropy for glycine is 6.7 cal/K. Both numbers are in close agreement with recent estimates made by entirely different methods, suggesting a promising degree of consistency between data obtained from disparate sources. In addition, a quantitative analysis of the folding free energy indicates that the unfavorable backbone entropy for each of the proteins is balanced predominantly by favorable backbone interactions. Finally, because the binding of flexible peptides to receptors is physically similar to folding, the free energy function should, in principle, be equally applicable to flexible docking. By combining atomic contact energies, electrostatics, and sequence-dependent backbone entropy, we calculated a priori the free energy changes associated with the binding of four different peptides to HLA-A2, 1 MHC molecule and found agreement with experiment to within 10% without parameter adjustment.

Determination of atomic desolvation energies from the structures of crystallized proteins.

We estimated effective atomic contact energies (ACE), the desolvation free energies required to transfer atoms from water to a protein's interior, using an adaptation of a method introduced by S. Miyazawa and R. L. Jernigan. The energies were obtained for 18 different atom types, which were resolved on the basis of the way their properties cluster in the 20 common amino acids. In addition to providing information on atoms at the highest resolution compatible with the amount and quality of data currently available, the method itself has several new features, including its reference state, the random crystal structure, which removes compositional bias, and a scaling factor that makes contact energies quantitatively comparable with experimentally measured energies. The high level of resolution, the explicit accounting of the local properties of protein interiors during determination of the energies, and the very high computational efficiency with which they can be assigned during any computation, should make the results presented here widely applicable. First we used ACE to calculate the free energies of transferring side-chains from protein interior into water. A comparison of the results thus obtained with the measured free energies of transferring side-chains from n-octanol to water, indicates that the magnitude of protein to water transfer free energies for hydrophobic side-chains is larger than that of n-octanol to water transfer free energies. The difference is consistent with observations made by D. Shortle and co-workers, who measured differential free energies of protein unfolding for site-specific mutants in which Ala or Gly was substituted for various hydrophobic side-chains. A direct comparison (calculated versus observed free energy differences) with those experiments finds slopes of 1.15 and 1.13 for Gly and Ala substitutions, respectively. Finally we compared calculated and observed binding free energies of nine protease-inhibitor complexes. This requires a full free energy function, which is created by adding direct electrostatic interactions and an appropriate entropic component to the solvation free energy term. The calculated free energies are typically within 10% of the observed values. Taken collectively, these results suggest that ACE should provide a reasonably accurate and rapidly evaluatable solvation component of free energy, and should thus make accessible a range of docking, design and protein folding calculations that would otherwise be difficult to perform.

Computational determination of side chain specificity for pockets in class I MHC molecules.

We show that a rapidly executable computational procedure provides the basis for a predictive understanding of antigenic peptide side chain specificity, for binding to class I major histocompatibility complex (MHC) molecules. The procedure consists of a combined search to identify the joint conformations of peptide side chains and side chains comprising the MHC pocket, followed by conformational selection, using a target function, based on solvation energies and modified electrostatic energies. The method was applied to the B pocket region of five MHC molecules, which were chosen to encompass the full range of specificities displayed by anchors at peptide position 2. These were a medium hydrophobic residue (Leu or Met) for HLA-A*0201, a basic residue (Arg or Lys) for HLA-B*2705; a small hydrophobic residue (Val) for HLA-A*6801, an acidic residue (Glu) for HLA-B*4001 and a bulky residue (Tyr) for H-2K(d). The observed anchors are correctly predicted in each case. The agreement for HLA-B40 and H-2K(d) is especially promising, since their structures have not yet been determined experimentally. Because the experimental determination of motifs by elution is difficult and these calculations take only hours on a high speed workstation, the results open the possibility of routine determination of motifs computationally.

Molecular analysis of a heteroclitic T cell response to the immunodominant epitope of sperm whale myoglobin. Implications for peptide partial agonists.

We have investigated the molecular basis for binding and Ag presentation of an immunodominant Th cell determinant of sperm whale myoglobin, a prototype amphipathic helical structure in the native protein. A series of peptides with three different substitutions at each position were evaluated for binding to the class II MHC molecule I-Ad and for activation of two T cell clones with distinct fine specificity, to determine the role of each residue. The assignment of MHC binding and TCR binding residues is consistent with a peptide bound as a twisted beta-strand, with 130 degrees twist similar to that of the influenza hemagglutinin peptide crystallized in the groove of HLA-DR1. This twist gives the peptide amphipathicity, with a periodicity similar to an alpha-helix without its being a helix. Two substituted peptides were discovered to be heteroclitic, but by different molecular mechanisms, one involving gain of a favorable residue and one involving loss of an unfavorable one. Complexes of both peptides with I-Ad had enormously higher affinity for the TCR, but peptide affinity for the MHC molecule was not increased, such that the wild-type peptide acted as a partial agonist and inhibited the response to the heteroclitic ones. Moreover, the magnitude of response was elevated in a way that could not be mimicked by the wild-type peptide even at higher concentration. These results suggest a TCR dwell time requirement for optimal signal transduction that may help explain the mechanism of partial agonism.

Periodic variation in side-chain polarities of T-cell antigenic peptides correlates with their structure and activity.

We present an analysis that synthesizes information on the sequence, structure, and motifs of antigenic peptides, which previously appeared to be in conflict. Fourier analysis of T-cell antigenic peptides indicates a periodic variation in amino acid polarities of 3-3.6 residues per period, suggesting an amphipathic alpha-helical structure. However, the diffraction patterns of major histocompatibility complex (MHC) molecules indicate that their ligands are in an extended non-alpha-helical conformation. We present two mutually consistent structural explanations for the source of the alpha-helical periodicity, based on an observation that the side chains of MHC-bound peptides generally partition with hydrophobic (hydrophilic) side chains pointing into (out of) the cleft. First, an analysis of haplotype-dependent peptide motifs indicates that the locations of their defining residues tend to force a period 3-4 variation in hydrophobicity along the peptide sequence, in a manner consistent with the spacing of pockets in the MHC. Second, recent crystallographic determination of the structure of a peptide bound to a class II MHC molecule reveals an extended but regularly twisted peptide with a rotation angle of about 130 degrees. We show that similar structures with rotation angles of 100-130 degrees are energetically acceptable and also span the length of the MHC cleft. These results provide a sound physical chemical and structural basis for the existence of a haplotype-independent antigenic motif which can be particularly important in limiting the search time for antigenic peptides.

Graphical representations of the class I MHC cleft.

We describe computer graphics and computer aided manufacture of three-dimensional models designed specifically to elucidate the cleft in the class I human leukocyte antigen. The models evolve from computer graphical representations and provide a geometrically and chemically concise and detailed view of the antigen binding site. The techniques provide a new approach to representations of binding sites. The model provides sufficient detail to support binding specificities analysis of active sites involved in protein and DNA binding.

Characterization of a helper T cell epitope recognized by mice of a low responder major histocompatibility type.

Most known helper T cell (Th) epitopes studied have naturally been immunodominant epitopes recognized by T cells from animals of high responder major histocompatibility complex (MHC) haplotype. We have previously found that most such immunodominant Th epitopes tend to be amphipathic alpha helices, that is, helices with hydrophobic residues on one side and hydrophilic residues on the other, and the corresponding peptide can usually elicit a response to the native protein. However, very few epitopes seen by MHC low responder T cells have been identified. Within the CNBr fragment of residues 1-55 of sperm whale myoglobin (SwMb), a Th epitope is known to exist that stimulates T cells from low responder H-2k mice, but it has not yet been localized to a length of 8-12 residues, the usual length of a Th epitope. To determine whether this low responder epitope would have similar properties, we located it using 10 evenly overlapping 15-residue peptides that span the region. Analysis of this region by the computer program predicted the site covered by two peptides (residues 26-40 and 31-45 which overlap by 10 residues) to be the most likely site for a Th epitope. Of the 10 peptides tested experimentally, only one peptide (residues 26-40) was able to stimulate two low responder Th clones that are specific for the 1-55 region. The peptide was able to prime T cells of low responder B10.BR mice in vivo for in vitro response to the native SwMb as well as to the peptide fragment of residues 1-55. Immunization of low responder mice with SwMb showed that, of the 10 overlapping peptides, the major site of response within the 1-55 region is to the identified peptide. Finally, an extended peptide of residues 24-42 was made to increase the amphipathic score. This extended peptide induced greater proliferation of the clones. Thus, this low responder epitope has properties similar to those of immunodominant epitopes recognized by high responders.

Identification of T-cell epitopes and use in construction of synthetic vaccines.

The T cell is central to the immune system response to foreign antigens, and understanding the mechanism of T cell response to antigen is crucial for vaccine development. Short subpeptides of foreign antigen can prime the T cells to respond to the whole antigen, in some cases as well as or better than immunization with the whole antigen itself. Antigenic sites located first in the murine model are also antigenic in the human, suggesting that the structural features of antigenic sites are species-independent. The amphipathic helix hypothesis has proven useful in developing an algorithm that has successfully located immunodominant sites in important proteins, thus reducing substantially the experimental time and effort required to locate those sites. Other algorithms have also been used successfully, but in all cases there are proven T-cell sites not accounted for by the algorithm. A data base showing T-cell response to collections of peptides uniformly distributed along protein antigens would be very useful in subsequent efforts to characterize the physical and chemical properties of T-cell antigenic sites.

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.

Synthetic peptides derived from IRBP induce EAU and EAP in Lewis rats.

In an earlier study we isolated three cyanogen bromide cleavage fragments of bovine IRBP that exhibited high levels of immunopathogenicity, producing inflammatory changes in the eyes (EAU) and pineal gland (EAP) of Lewis rats. These fragments have been localized within the IRBP sequence. In order to identify these putative immunopathogenic epitopes of IRBP, nine selected peptide sequences were synthesized and tested for the induction of disease in Lewis rats. Seven of the peptides were found inactive in producing disease while two closely related peptides, designated R4 (23-mer) and R9 (27-mer) were found to reproducibly induce EAU and EAP in immunized rats. No good correlation was found between the immunopathogenicity of the nine tested peptides and their amphipathicity: peptides R4 and R9 were not predicted to form strong amphipathic helices, while peptides selected for their high predicted helical amphipathicity were not immunopathogenic. EAU induced by peptides R4 and R9 was less severe and had a longer onset time than the disease induced by whole IRBP. In addition, the inflammatory changes induced by R4 and R9 in the posterior segment of the eye were less acute than those induced by whole IRBP and included granuloma formation and perivasculitis, features which are not generally seen in rats immunized with whole IRBP. Thus, the changes induced by R4 and R9 more closely resemble those which are characteristically found in human eyes affected by certain uveitic diseases than do changes produced by the intact protein.

An immunodominant epitope of the human immunodeficiency virus envelope glycoprotein gp160 recognized by class I major histocompatibility complex molecule-restricted murine cytotoxic T lymphocytes.

Because cytotoxic T lymphocytes (CTL) may be important for preventing direct cell-to-cell transmission of human immunodeficiency virus (HIV), the agent responsible for acquired immunodeficiency syndrome, we have begun to investigate the epitope specificity and immune response (Ir) gene control of anti-HIV CTL responses in experimental animals. Mice were infected with a recombinant vaccinia virus expressing the HIV gp160 envelope gene, and the primed lymphocytes were restimulated in vitro with a transfected histocompatible cell line expressing the same gene. Our results show that H-2d mice are CTL high responders and H-2k mice are low responders to the HIV gp160 envelope protein under these conditions. Moreover, the H-2d mice respond predominantly to a single immunodominant site represented by a 15-residue synthetic peptide conforming to the amphipathic alpha-helix model of T-cell epitopes and seen by CD4- CD8+ CTL in association with the Dd class I major histocompatibility complex (MHC) molecules. The facts that CTL responses were detected in the context of only one of four class I MHC molecules tested and that the response was limited predominantly to a single epitope indicate that the CTL repertoire elicited by the HIV envelope protein in association with murine class I MHC molecules may be very limited. In addition, this epitope occurs in a highly variable segment of the envelope protein. This puts constraints on the use of a single peptide sequence from this region in a vaccine, as such a vaccine would have to be polyvalent. Nevertheless, this same variability suggests that this region may be under selective pressure from human CTL, and therefore that this site may be immunodominant in humans as well as mice and so of clinical importance in vaccine development.

Some mathematical aspects of mapping DNA cosmids.

A number of experimental and mathematical problems must be solved before high resolution physical maps of mammalian chromosomes can be reliably determined. Such a map might consist of an ordered set of nonsequenced, overlapping DNA fragments 20,000-40,000 bases long, produced by digestion of a chromosome, using two restriction enzymes. Map construction requires assigning a signature to each fragment that differentiates it unambiguously from every other fragment, and then devising a computationally efficient algorithm that will provide a unique ordering of the fragments. In the first part of this paper we present a polynomial time algorithm that yields a unique map, and is largely independent of the method for assigning signatures. In the next section we analyze the distribution of lengths of restriction digest fragments and discuss the implications for the algorithm, including the expected number of map gaps. Finally, we discuss a specific method for assigning signatures proposed by Hans Lehrach, based on which of a panel of probes binds to a given fragment. In particular we examine the effects of fragment length heterogeneity on the theoretical optimum length and number of probes, and the extent to which false signatures might be obtained by nonspecific binding. We conclude that the Lehrach strategy is effective provided the number of probes is greater than or equal to 150, but that each fragment will need testing with at most 25 probes.

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)

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)

Helper T-cell antigenic site identification in the acquired immunodeficiency syndrome virus gp120 envelope protein and induction of immunity in mice to the native protein using a 16-residue synthetic peptide.

Much effort has been devoted to the analysis of antibodies to acquired immunodeficiency syndrome virus antigens, but no studies, to our knowledge, have defined antigenic sites of this virus that elicit T-cell immunity, even though such immunity is important in protection against many other viruses. T cells tend to recognize only a limited number of discrete sites on a protein antigen. Analysis of immunodominant helper T-cell sites has suggested that such sites tend to form amphipathic helices. An algorithm based on this model was used to identify two candidate T-cell sites, env T1 and env T2, in the envelope protein of human T-lymphotropic virus type IIIB that were conserved in other human immunodeficiency virus isolates. Corresponding peptides were synthesized and studied in genetically defined inbred and F1 mice for induction of lymph node proliferation. After immunization with a 426-residue recombinant envelope protein fragment, significant responses to native gp 120, as well as to each peptide, were observed in both F1 combinations studied. Conversely, immunization with env T1 peptide induced T-cell immunity to the native gp 120 envelope protein. The genetics of the response to env T1 peptide were further examined and revealed a significant response in three of four independent major histocompatibility haplotypes tested, an indication of high frequency responsiveness in the population. Identification of helper T-cell sites should facilitate development of a highly immunogenic, carrier-free vaccine that induces T-cell and B-cell immunity. The ability to elicit T-cell immunity to the native viral protein by immunization with a 16-residue peptide suggests that such sites represent potentially important components of an effective vaccine for acquired immunodeficiency syndrome.

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.

Construction of synthetic immunogen: use of new T-helper epitope on malaria circumsporozoite protein.

The circumsporozoite (CS) protein of Plasmodium falciparum is the focus of intense efforts to develop an antisporozoite malaria vaccine. Localization of sites for T-cell recognition on this molecule is critical for vaccine design. By using an algorithm designed to predict T-cell sites and a large panel of H-2 congenic mice, a major nonrepetitive T-cell site was located. When a synthetic peptide corresponding to this site was covalently linked to the major B-cell site on the molecule, an immunogen capable of eliciting a high-titer antibody response was formed. This peptide sequence could prime helper T cells for a secondary response to the intact CS protein. The new helper T-cell site is located outside the repetitive region of the CS protein and appears to be the immunodominant T site on the molecule. This approach should be useful in the rational design and construction of vaccines.