Bioinformatic requirements for protein database searching using predicted epitopes from disease-associated antibodies

We describe a new approach to identify proteins involved in disease pathogenesis. The technology, Epitope-Mediated Antigen Prediction (E-MAP), leverages the specificity of patients' immune responses to disease-relevant targets and requires no prior knowledge about the protein. E-MAP links pathologic antibodies of unknown specificity, isolated from patient sera, to their cognate antigens in the protein database. The E-MAP process first involves reconstruction of a predicted epitope using a peptide combinatorial library. We then search the protein database for closely matching amino acid sequences. Previously published attempts to identify unknown antibody targets in this manner have largely been unsuccessful for two reasons: 1) short predicted epitopes yield too many irrelevant matches from a database search and 2) the epitopes may not accurately represent the native antigen with sufficient fidelity. Using an in silico model, we demonstrate the critical threshold requirements for epitope length and epitope fidelity. We find that epitopes generally need to have at least seven amino acids, with an overall accuracy of >70% to the native protein, in order to correctly identify the protein in a nonredundant protein database search. We then confirmed these findings experimentally, using the predicted epitopes for four monoclonal antibodies. Since many predicted epitopes often fail to achieve the seven amino acid threshold, we demonstrate the efficacy of paired epitope searches. This is the first systematic analysis of the computational framework to make this approach viable, coupled with experimental validation.     

Linkage of Amino Acid Variation and Evolution of Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein (Subtype B) with Usage of the Second Receptor

To clarify the relationship between the amino acid variations of the gp120 of human immunodeficiency virus type 1 (HIV-1) and the chemokine receptors that are used as the second receptor for HIV, we evaluated amino acid site variation of gp120 between the X4 strains (use CXCR4) and the R5 strains (use CCR5) from 21 sequences of subtype B. Our analysis showed that residues 306 and 322 in the V3 loop and residue 440 in the C4 region were associated with usage of the second receptor. The polymorphism at residue 440 is clearly associated with the usage of the second receptor: The amino acid at position 440 was a basic amino acid in the R5 strains, and a nonbasic and smaller amino acid in the X4 strains, while the V3 loop of the X4 strains was more basic than that of the R5 strains. This suggests that residue 440 in the C4 region, which is close to the V3 loop in the three-dimensional structure, is critical in determining which second receptor is used. Analysis of codon frequency suggests that, in almost all cases, the difference at residue 440 between basic amino acids in the R5 strains and nonbasic amino acids in the X4 strains could be due to a single nucleotide change. These findings predict that the evolutionary changes in amino acid residue 440 may be correlated with evolutionary changes in the V3 loop. One possibility is that a change in electric charge at residue 440 compensates for a change in electric charge in the V3 loop. The amino acid polymorphism at position 440 can be useful to predict the cell tropism of a strain of HIV-1 subtype B.     

Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.

Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule.     

Comparative magnitude of cross-strain conservation of HIV variable loop neutralization epitopes

Although the sequence variable loops of the human immunodeficiency virus' (HIV-1) surface envelope glycoprotein (gp120) can exhibit good immunogenicity, characterizing conserved (invariant) cross-strain neutralization epitopes within these loops has proven difficult. We recently developed a method to derive sensitive and specific signature motifs for the three-dimensional (3D) shapes of the HIV-1 neutralization epitopes in the third variable (V3) loop of gp120 that are recognized by human monoclonal antibodies (mAbs). We used the signature motif method to estimate the conservation of these epitopes across circulating worldwide HIV-1 strains. The epitope targeted by the anti-V3 loop neutralizing mAb 3074 is present in 87% of circulating strains, distributed nearly evenly among all subtypes. The results for other anti-V3 Abs are: 3791, present in 63% of primarily non-B subtypes; 2219, present in 56% of strains across all subtypes; 2557, present in 52% across all subtypes; 447-52D, present in 11% of primarily subtype B strains; 537-10D, present in 9% of primarily subtype B strains; and 268-D, present in 5% of primarily subtype B strains. The estimates correlate with in vitro tests of these mAbs against diverse viral panels. The mAb 3074 thus targets an epitope that is nearly completely conserved among circulating HIV-1 strains, demonstrating the presence of an invariant structure hidden in the dynamic and sequence-variable V3 loop in gp120. Since some variable loop regions are naturally immunogenic, designing immunogens to mimic their conserved epitopes may be a promising vaccine discovery approach. Our results suggest one way to quantify and compare the magnitude of the conservation.     

Expression of meningococcal epitopes in LamB of Escherichia coli and the stimulation of serosubtype-specific antibody responses

The class 1 outer membrane protein (OMP), a major variable surface antigen of Neisseria meningitidis, is a component of novel meningococcal vaccines currently in field trials. Serological variants of the protein are also used to serosubtype meningococci. Most of the amino acid changes that give rise to antigenic variants of the protein occur in two variable regions (VR1 and VR2) that are thought to form loops on the cell surface. The polymerase chain reaction (PCR) was used to amplify the nucleotide sequences encoding VR1 and VR2 from the chromosomal DNA of N. meningitidis strain M1080. These were cloned in frame into the lamB gene of the Escherichia coli expression vector pAJC264. Whole-cell enzyme-linked immunosorbent assays (ELISAs), using monoclonal antibodies, and SDS PAGE confirmed that, upon induction, strains of E. coli carrying these constructs expressed hybrid LamB proteins containing the N. meningitidis surface loops. These strains were used to immunize rabbits and the resultant polyclonal antisera reacted specifically with the class 1 OMP of reference strain M1080 (P1.7). Immunogold labelling of meningococcal cells and whole-cell dot-blot analyses with these antisera showed that the variable epitopes were exposed on the cell surface and confirmed that this approach could be used to obtain serosubtype-specific antisera. The binding profiles of the antisera were determined from their reactions with overlapping synthetic peptides and their reactivity compared with that of relevant serosubtype-specific monoclonal antibodies. This approach was used successfully to raise antisera against two other class 1 OMP VR2s. A fourth antiserum raised against a VR2, including the P1.1 epitope, was not subtype specific.     

Characterization of two distinct major histocompatibility complex class I Kk-restricted T-cell epitopes within the influenza A/PR/8/34 virus hemagglutinin.

Cytotoxic T-lymphocyte (CTL) clones specific for the influenza A/PR/8/34 virus hemagglutinin (HA) were isolated by priming CBA mice with a recombinant vaccinia virus expressing the HA molecule. The epitopes recognized by two of these clones, which were CD8+, Kk restricted, and HA subtype specific, were defined by using a combination of recombinant vaccinia viruses expressing HA fragments and synthetic peptides. One epitope is in the HA1 subunit at residues 259 to 266 (numbering from the initiator methionine), amino acid sequence FEANGNLI, and the other epitope is in the HA2 subunit at residues 10 to 18 (numbering from the amino terminus of the HA2 subunit), sequence IEGGWTGMI. These two peptides are good candidates for naturally processed HA epitopes presented during influenza infection, as they are the same length (eight and nine residues) as other naturally processed viral peptides presented to CTL. A comparison of the sequences of these two new epitopes with those of the three previously published Kk-restricted T-cell epitopes showed some homology among all of the epitopes, suggesting a binding motif. In particular, an isoleucine residue at the carboxy-terminal end is present in all of the epitopes. On the basis of this homology, we predicted that the Kk-restricted epitope in influenza virus nucleoprotein, previously defined as residues 50 to 63, was contained within residues 50 to 57, sequence SDYEGRLI. This shorter peptide was found to sensitize target cells at a 200-fold lower concentration than did nucleoprotein residues 50 to 63 when tested with a CTL clone, confirming the alignment of Kk-restricted epitopes.     

Identification of variable region differences in Neisseria meningitidis class 3 protein sequences among five group B serotypes

Strains of Neisseria meningitidis express one of two porin proteins. These porins have been identified as the class 2 and class 3 proteins, and express serotype-specific epitopes. The gene for the class 3 protein was amplified by the polymerase chain reaction from the DNA of a serotype 4 strain as a 1025 bp fragment. The nucleotide sequence of this gene was determined and compared with two recently published sequences. On the basis of this comparison we have identified two major variable regions in the translated protein sequence, VR1 and VR2, that may be associated with serotype specificity. Three other variable regions were also identified. The sequences in the VR1 and VR2 regions from five additional group B N. meningitidis strains of serotypes 1, 4, 8, 12, and 15, all expressing class 3 proteins, were determined. The VR1 and VR2 regions were variable and were flanked by highly conserved regions among eight different class 3 sequences. These two variable regions of 15 and 9 amino acids are predicted to be in surface-exposed loops.     

Naturally Occurring Mutations within 39 Amino Acids in the Envelope Glycoprotein of Maedi-Visna Virus Alter the Neutralization Phenotype

Infectious molecular clones have been isolated from two maedi-visna virus (MVV) strains, one of which (KV1772kv72/67) is an antigenic escape mutant of the other (LV1-1KS1). To map the type-specific neutralization epitope, we constructed viruses containing chimeric envelope genes by using KV1772kv72/67 as a backbone and replacing various parts of the envelope gene with equivalent sequences from LV1-1KS1. The neutralization phenotype was found to map to a region in the envelope gene containing two deletions and four amino acid changes within 39 amino acids (positions 559 to 597 of Env). Serum obtained from a lamb infected with a chimeric virus, VR1, containing only the 39 amino acids from LV1-1KS1 in the KV1772kv72/67 backbone neutralized LV1-1KS1 but not KV1772kv72/67. The region in the envelope gene that we had thus shown to be involved in escape from neutralization was cloned into pGEX-3X expression vectors, and the resulting fusion peptides from both molecular clones were tested in immunoblots for reactivity with the KV1772kv72/67 and VR1 type-specific antisera. The type-specific KV1772kv72/67 antiserum reacted only with the fusion peptide from KV1772kv72/67 and not with that from LV1-1KS1, and the type-specific VR1 antiserum reacted only with the fusion peptide from LV1-1KS1 and not with that from KV1772kv72/67. Pepscan analysis showed that the region contained two linear epitopes, one of which was specific to each of the molecularly cloned viruses. This linear epitope was not bound by all type-specific neutralizing antisera, however, which indicates that it is not by itself the neutralization epitope but may be a part of it. These findings show that mutations within amino acids 559 to 597 in the envelope gene of MVV virus result in escape from neutralization. Furthermore, the region contains one or more parts of a discontinuous neutralization epitope.     

Rationally Targeted Mutations at the V1V2 Domain of the HIV-1 Envelope to Augment Virus Neutralization by Anti-V1V2 Monoclonal Antibodies

HIV-1 envelope glycoproteins (Env) are the only viral antigens present on the virus surface and serve as the key targets for virus-neutralizing antibodies. However, HIV-1 deploys multiple strategies to shield the vulnerable sites on its Env from neutralizing antibodies. The V1V2 domain located at the apex of the HIV-1 Env spike is known to encompass highly variable loops, but V1V2 also contains immunogenic conserved elements recognized by cross-reactive antibodies. This study evaluates human monoclonal antibodies (mAbs) against V2 epitopes which overlap with the conserved integrin α4β7-binding LDV/I motif, designated as the V2i (integrin) epitopes. We postulate that the V2i Abs have weak or no neutralizing activities because the V2i epitopes are often occluded from antibody recognition. To gain insights into the mechanisms of the V2i occlusion, we evaluated three elements at the distal end of the V1V2 domain shown in the structure of V2i epitope complexed with mAb 830A to be important for antibody recognition of the V2i epitope. Amino-acid substitutions at position 179 that restore the LDV/I motif had minimal effects on virus sensitivity to neutralization by most V2i mAbs. However, a charge change at position 153 in the V1 region significantly increased sensitivity of subtype C virus ZM109 to most V2i mAbs. Separately, a disulfide bond introduced to stabilize the hypervariable region of V2 loop also enhanced virus neutralization by some V2i mAbs, but the effects varied depending on the virus. These data demonstrate that multiple elements within the V1V2 domain act independently and in a virus-dependent fashion to govern the antibody recognition and accessibility of V2i epitopes, suggesting the need for multi-pronged strategies to counter the escape and the shielding mechanisms obstructing the V2i Abs from neutralizing HIV-1.     

Predictions of linear T-cell and B-cell epitopes in proteins encoded by HIV-1, HIV-2 and SIVMAC and the conservation of these sites between strains

An important consideration in the design of vaccines to prevent HIV-1 infection effective against different strains is the amino acid sequence conservation of antigenic determinants. Even one amino acid change can destroy the antigenicity of a site for the antibody or T-cell receptor. The comparisons of predicted T- and B-cell epitopes between human HIV-1, HIV-2 and monkey SIVMAC AIDS viruses are presented. The three major gene products (env, gag and pol) were examined. A number of epitopes were identical between strains of HIV-1. Our analysis highlights the problem of designing an effective HIV-1 and HIV-2 vaccine and also the problem of testing human vaccines in monkey models.     

epitopes immediately below the base of the V3 loop of gp120 as targets for the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals

Epitopes that drive the initial autologous neutralizing antibody response in HIV-1-infected individuals could provide insights for vaccine design. Although highly strain specific, these epitopes are immunogenic, vulnerable to antibody attack on infectious virus, and could be involved in the ontogeny of broadly neutralizing antibody responses. To delineate such epitopes, we used site-directed mutagenesis, autologous plasma samples, and autologous monoclonal antibodies to map the amino acid changes that led to escape from the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals. Additional mapping of the epitopes was accomplished by using alanine scanning mutagenesis. Escape in the two individuals occurred by different pathways, but the responses in both cases appeared to be directed against the same region of gp120. In total, three amino acid positions were identified that were independently associated with autologous neutralization. Positions 295 and 332 are located immediately before and after the N- and C-terminal cysteines of the V3 loop, respectively, the latter of which affected an N-linked glycan that was critical to the neutralization epitope. Position 415 affected an N-linked glycan at position 413 in the C terminus of V4 that might mask epitopes near the base of V3. All three sites lie in close proximity on a four-stranded antiparallel sheet on the outer domain of gp120. We conclude that a region just below the base of the V3 loop, near the coreceptor binding domain of gp120, can be a target for autologous neutralization.     

Analysis of murine B-cell epitopes on Eastern equine encephalitis virus glycoprotein E2

The Eastern equine encephalitis virus (EEEV) E2 protein is one of the main targets of the protective immune response against EEEV. Although some efforts have done to elaborate the structure and immune molecular basis of Alphaviruses E2 protein, the published data of EEEV E2 are limited. Preparation of EEEV E2 protein-specific antibodies and define MAbs-binding epitopes on E2 protein will be conductive to the antibody-based prophylactic and therapeutic and to the study on structure and function of EEEV E2 protein. In this study, 51 EEEV E2 protein-reactive monoclonal antibodies (MAbs) and antisera (polyclonal antibodies, PAbs) were prepared and characterized. By pepscan with MAbs and PAbs using enzyme-linked immunosorbent assay, we defined 18 murine linear B-cell epitopes. Seven peptide epitopes were recognized by both MAbs and PAbs, nine epitopes were only recognized by PAbs, and two epitopes were only recognized by MAbs. Among the epitopes recognized by MAbs, seven epitopes were found only in EEEV and two epitopes were found both in EEEV and Venezuelan equine encephalitis virus (VEEV). Four of the EEEV antigenic complex-specific epitopes were commonly held by EEEV subtypes I/II/III/IV (1-16aa, 248-259aa, 271-286aa, 321-336aa probably located in E2 domain A, domain B, domain C, domain C, respectively). The remaining three epitopes were EEEV type-specific epitopes: a subtype I-specific epitope at amino acids 108–119 (domain A), a subtype I/IV-specific epitope at amino acids 211–226 (domain B) and a subtype I/II/III-specific epitope at amino acids 231–246 (domain B). The two common epitopes of EEEV and VEEV were located at amino acids 131–146 and 241–256 (domain B). The generation of EEEV E2-specific MAbs with defined specificities and binding epitopes will inform the development of differential diagnostic approaches and structure study for EEEV and associated alphaviruses.     

Evaluation of diagnostic efficiency of the recombinant protein modeling immunodominant epitope V3 of envelope gp120 for immunoenzyme detection for HIV-1 infection antibodies

The third hypervariable domain V3 of the human immunodeficiency virus type 1 gpl20 envelope glycoprotein contains neutralizing epitopes and plays an important role in the diagnosis of HIV infection . Neutralizing antibodies bind to conserved epitope with sequence GPG of V3 loop. The effect of sequence variation on the antigenic properties of the V3 epitope gp120 was studied using five synthetic peptides. The amino acid sequence of the peptide corresponding to the V3 region gp120 of HIV-1 subtype C showed the highest immunoreactivity. The DNA fragment encoding V3-C region gp120 was synthesized by polymerase chain reaction and cloned into pET41b vector. The recombinant plasmid was expressed in the E. coli cells, and recombinant protein was purified using glutathione-S sepharose affinity chromatography. The serological activity of the recombinant protein was tested using ELISA and compared to activity of similar synthetic peptide. The results of this study showed that most immunoreactive agent was the amino acid sequence of V3 region gp120 of HIV-1 subtype C. The recombinant antigen comprising this sequence was more antigenic than synthetic peptide with the same sequence. The evaluation of this antigen shows that this protein is a good candidate for the immunoassay development.     

Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides

Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.     

Epitope peptides of influenza H3N2 virus neuraminidase gene designed by immunoinformatics

The virus surface protein neuraminidase (NA) is a main subtype-specific antigen in influenza type A viruses. Neuraminidase functions as an enzyme to break the bonds between hemagglutinin (HA) and sialic acid to release newly formed viruses from infected cells. In this study, NA genes from the H3N2 subtype virus were sequenced and NA proteins were screened for B-cell epitopes and assessed based on immunoinformatics. Based on this information, three peptides ES8, RR9, and WK7 (covering amino acid residues 221-228, 292-300, and 383-389, respectively) of the NA protein were selected and synthesized artificially. These peptides were used to immunize New Zealand rabbits subcutaneously to raise antisera. Results showed that these three peptides were capable of eliciting antibodies against H3N2 viruses in a specific and sensitive manner, detected in vitro by enzyme-linked immunosorbent assay. Furthermore, hemadsorption anti-releasing effects occurred in three antisera mixtures at a dilution of 1:40. Alignment using database software showed that amino acid residues in these three epitope peptides were substituted at specific sites in all the NAs sequenced in this study. We suggest that these NA epitope peptides might be used in conjunction with HA proteins as vaccine antigens.     

Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication

It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion.     

猪胸膜肺炎放线杆菌ApxIIA基因的序列分析及其B细胞表位预测

为了进一步研究猪胸膜肺炎放线杆菌(Actionobacillus pleuropneumoniae,App)ApxIIA基因的结构和功能,选取GenBank中6株不同App分离株,采用生物信息学方法对其ApxIIA基因及其编码氨基酸序列进行同源性比对,并选择其中AY232288.1菌株(湖北分离株)ApxIIA基因为研究对象,分析该基因所编码蛋白质的理化性质,并对其可能形成的二级结构及B细胞表位进行预测和分析.结果表明,6株不同App分离株ApxIIA基因核苷酸及氨基酸序列同源性分别为65.56﹪和88.42﹪,在AY232288.1菌株ApxIIA蛋白的肽链中,745～751和801～807区段可能是其B细胞表位优势区.本研究首次利用生物信息学方法对ApxIIA基因及其蛋白质结构进行了分析和预测,为ApxIIA基因功能的深入研究及APP多表位疫苗的设计奠定了基础.     

Mutation of human immunodeficiency virus type 1 at amino acid 585 on gp41 results in loss of killing by CD8+ A24-restricted cytotoxic T lymphocytes.

A human leukocyte antigen A24-restricted CD8+ cytotoxic T-cell clone specific for gp41 of human immunodeficiency virus type 1 was isolated from an infected individual. The epitope was localized to amino acids 584 to 591 (YLKDQQLL, NL43 env sequence) of gp41 by using a panel of recombinant vaccinia viruses that contain truncated env genes and synthetic peptides. The clone killed autologous B-lymphoblastoid cell lines pulsed with a synthetic peptide reflecting the sequence of the IIIB and MN strains. This clone, however, failed to kill target cells pulsed with the peptides that have a mutation from Lys to Arg or Gln at amino acid 585 which is present in some prototype human immunodeficiency virus type 1 strains, e.g., ADA, JFL, SC, ALA1, BAL1, SF2, VRF, SF33, and WMJ2. This finding that a mutation at amino acid 585 on gp41 results in nonrecognition by human leukocyte antigen A24-restricted CD8+ cytotoxic T lymphocytes suggests that antigenic variation at T-cell epitopes contributes to the failure of immune control of human immunodeficiency virus type 1 infections.     

Complete nucleotide sequence of the haemagglutinin gene from a human influenza virus of the Hong Kong subtype.

The complete nucleotide sequence has been determined for a cloned double-stranded DNA copy of the haemagglutinin gene from the human influenza strain A/NT/60/68/29C, a laboratory-isolated variant of A/NT/60/68, an early strain of the Hong Kong subtype. The gene is 1765 nucleotides long and contains information sufficient to code for a protein of 566 amino acids, which includes a hydrophobic leader peptide (16 residues), HA1 (328), HA2 (221) and an arginine residue which joins the HA subunits. Comparison of the predicted amino acid sequence for 29C haemagglutinin with protein sequence data available for HA from other influenza strains shows that no potential coding information is lost by processing of the mRNA. A comparison of the amino acid sequences predicted from the gene sequences for 29C and fowl plague virus haemagglutinins, (1) indicates the extent to which changes can occur in the primary sequence of different regions of the protein, while maintaining essential structure and function.