Computer Simulations to Identify in Polyproteins of FMDV OK1 and A12 Strains Putative Nonapeptides with Amino Acid Motifs for Binding to BoLA Class I A11 and A20 Haplotype Molecules

The computer program “Findpatterns” was used to search FMDV- (OK1 and A12 strains) coded structural and nonstructural proteins for the availability of putative proteasome-generated nonapeptides with motifs reported for BoLA class I A11 and A20 haplotypes. These BoLA class I A11 and A20 nonapeptide motifs are identical to motifs of nonapeptides that interact with the peptide binding grooves of HLA class I B35 and B27 haplotypes, respectively. The computer findpattern program was used to analyze the FMDV-coded polyproteins for proteolytically cleavable nonapeptides with motifs for binding to the peptide binding grooves of BoLA class I A11 or 20 haplotypes. The computer simulations revealed that FMDV-infected cells (keratinocytes and antigen presenting cells. e.g., dendritic Langerhans cells in bovines) may be able to present viral nonapeptides to CD8⁺ cytolytic T cells (CTLs) in a BoLA-restricted manner. The role of the cellular arm of the immune response i n the protection of bovines against FMDV is not known. Thus, the present computer analysis may encourage further experiments to develop a new generation of FMDV nonapeptide vaccines to stimulate the anti-FMDV cytolytic T cell response in bovine so this would complement the humoral immune response achieved by immunization with the inactivated virus vaccine. This revised version was published online in July 2006 with corrections to the Cover Date.     

Computer simulations to predict the availability of peptides with known HLA class I motifs generated by proteolysis of dengue fever virus (DFV) type 1 structural and nonstructural proteins in infected cells

Cytotoxic T cells that recognize dengue fever viral (DFV) peptides were reported. To predict the cleavage pattern of DFV proteins by cytoplasmic proteasomes into nonapeptides with motifs fitting known HLA class I molecules, the computer program Findpatterns was used. In this study the combined amino acid motifs for proteolytic cleavages and the HLA class I haplotype-restricted peptides were analyzed. It was noted that putative peptides with motifs of HLA A2 and A68 were abundant compared with nonapeptides with motifs HLA A24, B8, B35, and B53. The possible interpretation of the computer analysis in explaining the cellular immune response in endogenous populations of endemic DF is discussed.     

Computer prediction of antigenic and topogenic domains in HSV-1 and HSV-2 glycoprotein B (gB)

The envelope glycoprotein B (gB) coded for by the herpes simplex virus type 1 (HSV-1) U_L27 gene is similar to the amino acid (aa) sequence of the gB coded by a homologous gene in HSV-2 DNA. The putative antigenic domains in HSV-1 and HSV-2 gB glycoproteins were analyzed on a comparative basis by suitable computer programs, which allowed the prediction of putative antigenic and topogenic domains. The computer-derived domains were compared to experimentally reported antigenic domains in HSV-1 gB glycoprotein. The computer-predicted antigenic domains in the HSV-1 gB glycoprotein matched well with the reported experimentally derived antigenic domains. The aa sequence of antigenic domain 1 was noted to resemble the amino acid sequence in ApoE that is involved in the attachment of this protein to LDL receptors. The clusters of hydrophobic aa domains are conserved in the two viral glycoproteins and are signals for transfer of the viral proteins through the cellular membrane.     

HIV-1 proteins in infected cells determine the presentation of viral peptides by HLA class I and class II molecules and the nature of the cellular and humoral antiviral immune responses—A review

The goals of molecular virology and immunology during the second half of the 20th century have been to provide the conceptual approaches and the tools for the development of safe and efficient virus vaccines for the human population. The success of the vaccination approach to prevent virus epidemics was attributed to the ability of inactivated and live virus vaccines to induce a humoral immune response and to produce antiviral neutralizing antibodies in the vaccinees. The successful development of antiviral vaccines and their application to most of the human population led to a marked decrease in virus epidemics around the globe. Despite this remarkable achievement, the developing epidemics of HIV-caused AIDS (accompanied by activation of latent herpesviruses in AIDS patients), epidemics of Dengue fever, and infections with respiratory syncytial virus may indicate that conventional approaches to the development of virus vaccines that induce antiviral humoral responses may not suffice. This may indicate that virus vaccines that induce a cellular immune response, leading to the destruction of virus-infected cells by CD8⁺ cytotoxic T cells (CTLs), may be needed. Antiviral CD8⁺ CTLs are induced by viral peptides presented within the peptide binding grooves of HLA class I molecules present on the surface of infected cells. Studies in the last decade provided an insight into the presentation of viral peptides by HLA class I molecules to CD8⁺ T cells. These studies are here reviewed, together with a review of the molecular events of virus replication, to obtain an overview of how viral peptides associate with the HLA class I molecules. A similar review is provided on the molecular pathway by which viral proteins, used as subunit vaccines or inactivated virus particles, are taken up by endosomes in the endosome pathway and are processed by proteolytic enzymes into peptides that interact with HLA class II molecules during their transport to the plasma membrane of antigen-presenting cells. Such peptides are identified by T-cell receptors present on the plasma membrane of CD4⁺ T helper cells. The need to develop viral synthetic peptides that will have the correct amino acid motifs for binding to HLA class I A, B, and C haplotypes is reviewed.The development of HIV vaccines that will stimulate, in an uninfected individual, the humoral (antibody) and cellular (CTL) immune defenses against HIV and HIV-infected cells, respectively, and may lead to protection from primary HIV infection are discussed. The need to eliminate the release of HIV virions from infected cells introduced by an infected donor to an uninfected recipient may require both the humoral and cellular immune responses. However, such CTLs may fail to identify HIV-infected cells with integrated HIV proviral DNA that do not express viral genes and proteins. Based on reported results on the immunization of monkeys with uninfected cells, which prevented infection with SIV grown in the same type of cells, it may be possible to consider immunization of specific human populations against HLA haplotypes prevalent in HIV-infected donors. Since HIV virions may carry the HLA class I molecules present in the infected donors' cells, synthesis of CTLs to the mutated amino acid sequence in peptide binding grooves of the foreign HLA haplotypes may induce anti-HLA CTLs in the immunized individual, which may destroy HIV-infected, virus synthesizing donor cells, as well as donor cells containing latent proviral DNA. Such anti-foreign HLA CTLs may prevent the release of virions from the infecting donor's cells. The importance of HLA haplotypes for protection against HIV will be discussed.Dedicated to the memory of Dr. Albert Sabin (1906–1993).     

Putative antigenic domains in glycoprotein G of rabies virus: Is the RGK sequence involved in virus adsorption to cellular receptors?

Computer analysis of rabies virus glycoprotein G provides a means for identification of functional domains in the viral glycoprotein. The computer analysis suggested 22 putative antigenic domains, of which three are RG-containing amino acid sequences that might be involved in the binding of rabies virions to cellular receptors. Synthetic RG peptides may be able to interfere with rabies virus adsorption to cellular receptors.     

Human immunodeficiency virus type 1 gp120 C5 region mimics the HLA class I α1 peptide-binding domain

Molecular mimicry of major histocompatibility (MHC) antigens by viral glycoproteins has been suggested as one of the possible mechanisms of induction of an autoimmune response by human immunodeficiency viruses. A monoclonal antibody (M38) was previously shown to bind to both human immunodeficiency virus type 1 (HIV-1) gp120 and β-2 microglobulin-free HLA class I heavy chains encoded by an HLA C allele. Using HLA C recombinant proteins and synthetic peptides, the M38 class I binding site was mapped to a stretch of 44 amino acids of the al domain. The amino acid residues recognized are clustered in two non-contiguous regions at positions 66-69 (KYKR) and 79-82 (RKLR) shared by almost all HLA C alleles. On HIV-1 gp120, M38 binds to two non-contiguous sequences (KYK and KAKR) at positions 490-492 and 505-508 located at the edges of a large hydrophobic region that is apparently involved in binding the transmembrane glycoprotein gp41. The C-terminal gp120 M38-reactive region (KAKR) lies within the immunodominant sequence APTKAKRRVVQREKR, against which the majority of HIV-infected individuals produce antibodies. The results indicate that a functionally important region of HIV-1 gp120 shares similar amino acid sequence motifs with the antigen recognition site of most HLA class I C alleles. The molecular mimicry may be the basis for autoimmune responses in HIV infection.     

Dengue fever virus and Japanese encephalitis virus synthetic peptides,with motifs to fit HLA class I haplotypes prevalent in human populations in endemic regions,can be used for application to skin Langerhans cells to prime antiviral CD8+ cytotoxic T cells (CTLs)—A novel approach to the protection of humans

Flaviviruses were reported to induce CD8⁺ cytotoxic T cells in infected individuals, indicating that nonapeptides, proteolytic cleavage products of the viral precursor protein, enter the endoplasmic reticulum in infected cells and interact with HLA class I molecules. The assembled HLA class I molecules are transported to the plasma membrane and prime CD8⁺ T cells. Current knowledge of the interaction of viral peptides with HLA molecules is reviewed. Based on this review, an idea is presented to use synthetic flavivirus peptides with an amino acid motif to fit with the HLA class I peptide binding group of HLA haplotypes prevalent in a given population in an endemic area. These synthetic viral peptides may be introduced into the human skin using a lotion containing the peptides (Peplotion) together with substances capable of enhancing the penetration of these peptides into the skin to reach Langerhans cells. The peptide-treated Langerhans cells, professional antigen-presenting cells, may bind the synthetic viral peptides by their HLA class I peptide-binding grooves. Antigens carrying Langerhans cells are able to migrate and induce the cellular immune response in the lymph nodes. This approach to the priming of antiviral CD8⁺ cytotoxic T cells may provide cellular immune protection from flavivirus infection without inducing the humoral immune response, which can lead to the shock syndrome in Dengue fever patients. To be able to develop anti-Dengue virus synthetic peptides for populations with different HLA class I haplotypes, it is necessary to develop computational studies to design HLA class I Dengue virus synthetic peptides with motifs to fit the HLA haplotypes of the population living in an endemic region for Dengue fever. Experiments to study Dengue virus and Japanese encephalitis peptides vaccines and their effectiveness in protection against Dengue fever and Japanese encephalitis are needed. The development of human antiviral vaccines for application of viral peptides in a lotion to human skin (Peplotion) may be useful and affordable for populations of developing countries.     

Computer simulations to predict the availability of peptides with known HLA class I motifs possibly generated by proteolysis of HIV-1 proteins in infected cells

Cytotoxic T cells that recognize HIV-1 peptides generated from all viral proteins were reported. To predict the cleavage pattern of HIV-1 proteins by cytoplasmic proteasomes into peptides with motifs fitting known HLA class I molecules, the computer program Findpatterns was used. In this paper the combined amino acids patterns for proteolytic cleavages and the HLA class I haplotype-restricted peptides motifs are studied. It was noted that peptides with motifs of HLA class I A2 and A68 were abundant compared with HLA class 5B2, B8, B53, and B35.     

Need for cellular and humoral immune responses in bovines to ensure protection from foot-and-mouth disease virus (FMDV)—A point of view

The published studies on immunization of experimental animals, cattle, and sheep with synthetic peptides containing the antigenic domains in FMDV structural protein VP1 were analyzed. The results obtained with various FMDV synthetic peptides designed to stimulate the humoral immune response in bovines were compared to the current knowledge on MHC class I and class II, and the properties of the peptide binding grooves in each of them. X-ray crystallography of MHC class I proteins provided the three-dimensional structure of the peptide binding groove and led to the isolation and identification of self and viral peptides that naturally associate with the peptide binding grooves of both types of MHC and HLA molecules. The available knowledge of the amino acid motifs in MHC and HLA class I-bound viral peptides priming the CD8⁺ cytotoxic T cell responses must be coupled with the understanding of the three-dimensional structure of BoLA class I. This would aid in the development of an experimental approach to induce bovine anti-FMDV CD8⁺ cytotoxic cells to complement the humoral immune response to FMDV, which is currently achieved by a killed virus vaccine and, at the experimental level, by a peptide vaccine. Stimulation of both cellular and humoral immune responses against FMDV in cattle may reduce the risk of disease and virus shedding.     

Computer predictions of antigenic domains in human immunodeficiency virus-1 envelope glycoprotein: Comparison with reported experimental data

Computer analyses of amino acid sequences in the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein revealed that antigenic domains in the viral protein can be predicted on the basis of the physical properties of amino acids in the polypeptide chain. Relatively high values of surface probability, flexibility, and hydrophilicity were used as markers for domains of putative antigenicity. Comparison of the computer-predicted antigenic domains in the HIV-1 envelope with those reported experimentally indicate that computer analyses are able to predict antigenic domains. This study shows the usefulness of computer programs for the prediction of the antigenic domains in the HIV-1 envelope protein.Requests for reprints should be addressed to Yechiel Becker, Department of Molecular Virology, Faculty of Medicine, Hebrew University, P.O. Box 1172, Jerusalem, Israel.     

Computer predictions of antigenic domains in herpes simplex virus types 1 and 2 glycoprotein D as compared with experimentally proven domains

The primary amino-acid sequence of the glycoprotein D (gD) of herpes simplex virus types 1 and 2 (HSV-1, HSV-2) was analyzed by computer programs that provided values for hydrophilicity, surface probability, flexibility, and antigenicity, as well as the secondary structure conformation. Putative antigenic domains with a high hydrophilicity, surface probability, and antigenicity index were determined and compared with the reported antigenic domains in HSV-1 and HSV-2 gD protein based on experimental data. The major experimentally proven antigenic domains were detected by the computer analyses. Additional putative antigenic domains with potential for the synthesis of antigenic viral peptides were determined.     

Functional analysis of avian class I (BFIV) glycoproteins by epitope tagging and mutagenesis in vitro

Similarities between the physical structures of avian and mammalian major histocompatibility complex (MHC) class I glycoproteins have been proposed based on comparative alignment of their amino acid sequences. To investigate the physical structure of the chicken class I glycoprotein, we cloned the cDNA representing the BFIV locus of the B21 haplotype. A unique, chimeric class I glycoprotein was constructed by incorporating an epitope tag (FLAG) at the N terminus. Monoclonal antibodies to the FLAG epitope served to monitor cellsurface expression for functional analysis of the BFIV21 class I glycoprotein. The chimeric class I glycoprotein was expressed in target cells using an avian leukosis virus (ALV)-derived retrovirus vector (RCASBP). The presence of the FLAG epitope did not interfere with either alloantibody recognition or cytotoxic T lymphocyte interaction. Functional analysis employing site-directed mutagenesis identified BF amino acid residues forming serologic epitopes as well as residues important in antigen presentation to ALV-induced cytotoxic T lymphocytes. BF residues 78 and 81, corresponding to HLA 79 and 82, form an antibody epitope with a slight effect on ALV antigen presentation, consistent with their predicted orientation based on the HLA-A2 crystal structure. Alignment of the BFIV21 sequence with previously published BFIV sequences revealed polymorphisms at position 34 (HLA 34), a monomorphic residue in HLA and H-2. Residue 34 is located in pocket B and is predicted to contact the main-chain carbon of peptides bound in HLA-A2. A site-directed substitution in BFIV residue 34 dramatically alters ALV antigen presentation by the BFIV21 class I glycoprotein. These data indicate that the physical molecular structure of the chicken MHC class I glycoprotein is similar to HLA.     

An analysis of the role of skin Langerhans cells (LC) in the cytoplasmic processing of HIV-1 peptides after “Peplotion” transepidermal transfer and HLA class I presentation to CD8+ CTLs—An approach to immunization of humans

Skin Langerhans cells (LC) are antigen-presenting cells capable of expressing MHC class I and class II molecules on the plasma membrane. This molecular activity was reviewed to combine the knowledge of peptide presentation by MHC and HLA class I and class II molecules to prime CD8⁺ cytotoxic T cells (CTLs) and CD4⁺ T helper cells, respectively. The possible utilization of the skin dendritic cells for the development of antiviral CTLs and antibodies by synthetic peptides modeled according to the motifs of peptides that naturally interact with the peptide binding grooves of the various HLA haplotypes is discussed and evaluated. It may be possible that the introduction of synthetic viral peptides with motifs to fit the HLA class I haplotypes of a human population to the skin dendritic cells will prime selectively the cellular or the humoral immune responses. This approach may provide a new vaccination technique that applies synthetic virus peptides as vaccines for the immunization of humans. The neuropeptide CGRP interacts with LC and modulates antigen presentation.     

Computer analysis of antigenic domains and RGD-like sequences (RGWG) in the E glycoprotein of flaviviruses: An approach to vaccine development

Antigenic domains and RGD-like sequences in the E glycoprotein of the flaviviruses Japanese encephalitis virus, yellow fever virus, West Nile virus, dengue type 4 virus, and tick-borne encephalitis virus were analyzed by computer programs that provide information on the physical properties of the polypeptides. The use of computer programs for the development of vaccines based on the synthesis of antigenic peptides is discussed. Synthetic viral peptides are proposed to be used for topical application so as to interfere with the virus-cell interaction. Viral peptides with antigenic epitopes to protect against dengue virus infection without enhancing pathogenesis may also be developed on the basis of the computer analysis.     

Exploring myelin basic protein for HLA class I-binding sequences

In view of the increasing evidence of the involvement of CD8⁺ T cells in the pathogenesis of multiple sclerosis (MS), we have scanned the sequence of the myelin basic protein (MBP), using 162 overlapping nonapeptides, for HLA-class I binding sites. Peptide binding was measured using the recently reported HLA class I α-chain-refolding assay, and the following HLA allelic products were analyzed: HLA-A2 (*0201, *0204), B27 (*2705), B35, B51, and B62. A considerable number of binding peptides were distinguished for each of the allelic products tested. In addition, three interesting points emerged. The first was the identification of several binding peptides which did not contain the known anchor motifs. The second was the evidence that several peptides showed a promiscuous binding profile, being able to bind to different HLA class I molecules that were either allelic or non allelic. The third was that in several cases two consecutive peptides could bind to the same HLA molecule.     

High‐affinity human leucocyte antigen class I binding variola‐derived peptides induce CD4+ T cell responses more than 30 years post‐vaccinia virus vaccination

Interferon‐γ secreting T lymphocytes against pox virus‐derived synthetic 9‐mer peptides were tested by enzyme‐linked immunospot in peripheral blood of individuals vaccinated with vaccinia virus more than 30 years ago. The peptides were characterized biochemically as high‐affinity human leucocyte antigen (HLA) class I binders (K_D ≤ 5 nM). However, five of the individuals tested did not show typical CD8⁺ T cell‐mediated HLA class I‐restricted responses. Instead, these donors showed CD4⁺ T cell‐dependent responses against four of a total of eight antigenic 9‐mer peptides discovered recently by our group. These latter responses were blocked specifically in the presence of anti‐HLA class II antibody. We conclude that long‐lived memory responses against pox virus‐derived 9‐mer peptides, with high binding affinity for HLA class I molecules, are mediated in some cases by CD4⁺ T cells and apparently restricted by HLA class II molecules.     

Vaccination and infection as causative factors in Japanese patients with Rasmussen syndrome: molecular mimicry and HLA class I

Rasmussen syndrome is an intractable epilepsy with a putative causal relation with cellular and humoral autoimmunity. Almost half of the patients have some preceding causative factors, with infections found in 38.2%, vaccinations in 5.9% and head trauma in 8.9% of Japanese patients. In a patient with seizure onset after influenza A infections, cross-reaction of the patient's lymphocytes with GluR epsilon 2 and influenza vaccine components was demonstrated by lymphocyte stimulation test. Database analyses revealed that influenza A virus hemagglutinin and GluR epsilon 2 molecules contain peptides with the patient's HLA class I binding motif (HLA - A*0201). The relative risks of HLA class I genotypes for Rasmussen syndrome are 6.1 (A*2402), 6.4 (A*0201), 6.3 (A*2601) and 11.4 (B*4601). The relative risks of HLA class I-A and B haplotypes are infinity (A*2601 + B*5401), 21.1 (A*2402 + B*1501), 13.3 (A*2402 + B*4801) and 5.1 (A*2402 + B*5201). Some alleles and haplotypes of HLA class I may be the risk factors in Japanese patients. Cross-reactivity of cytotoxic T lymphocytes may contribute to the processes leading from infection to the involvement of CNS.     

Human leukocyte antigen-A, -B and -C alleles and human leukocyte antigen haplotypes in Turkey: relationship to other populations

In this study, we present, for the first time, human leukocyte antigen (HLA) class I allele and haplotype frequencies at the DNA level in a sample of 142 donors from Turkey. HLA typing was performed by medium-to-high resolution polymerase chain reaction sequence-specific oligonucleotide probes method. The most frequent HLA alleles at class I locus were A*0201(0.257), -B*35(0.204) and -Cw*04(0.173). A*0201-B*35-Cw*04(0.056) was the most common three-locus haplotype. Allele and haplotype frequency comparisons and neighbour-joining dendrograms, constructed using DA genetic distances and correspondence analysis using HLA-A, -B and -C, and -DRB1 allele frequencies, revealed similarities with other Mediterranean and European populations, but not with Mongol populations. These results agree with previous studies and confirm that the present day Turkish population is genetically more similar to its geographic neighbours than its historical neighbours in central Asia. The comprehensive HLA data on the Turkish population at the DNA level including up to six-locus putative haplotypes generated in this study will be useful for further studies.