Identification of residues involved in polymorphic antibody binding epitopes on HLA-DR molecules.

Based on previous studies it was predicted that amino acids 4 or 25 of the DR4 beta 1 and DR7 beta 1 chains are involved in polymorphic antibody binding epitopes on DR4 or DR7 molecules. These predictions were tested by analyzing monoclonal antibody (mAb) binding to transfectants expressing mutant DR4 beta 1 or DR7 beta 1 chains with single amino acid substitutions at positions 4 or 25. Antibody binding to transfectants expressing additional DR4/7 beta 1 hybrids was also analyzed to assess further the contributions of four segments of the DR4 beta 1 or DR7 beta 1 chains: amino acids 1-20, 21-40, 41-97, and the beta 2 domain. Single amino acid substitutions at positions 4 and 25 of the DR4 beta 1 chain or DR7 beta 1 chain eliminate binding of several mAb to DR4 or DR7 molecules, documenting that these residues are involved in antibody epitopes. However, the data with the hybrid DR4/7 beta 1 chains indicate that some of these epitopes require contributions from both segments 1-20 and 21-40 of these DR beta chains, whereas other epitopes can be generated by placing the appropriate segment in the context of the other DR beta chain. In addition, the data with other mAb indicate that their epitopes are determined primarily by sequences within the 41-97 segment or in the beta 2 domain.     

Role of strong anchor residues in effective binding of 10-MER and 11-MER peptides to HLA-A2402 molecules.

Analysis of the structural requirements for the interaction of antigenic peptides with HLA-A24 molecules are very important for studies of T cell recognition of various antigens, because HLA-A24 (A^*2402) is most common HLA-A allele in the world, especially in Oriental population. In order to precisely investigate the interaction of peptides with HLA-A24 molecules beyond previous analysis of self-peptides eluted from HLA-A24 molecules, we examined the A^*2402 interaction of 172 chemically synthesized 8-mer to 11-mer peptides carrying two residues (Try and Phe) at P2 and four residues (Phe, Trp, Leu and Ile) at their C-terminus by the use of stabilization assay. The results were statistically analyzed to assess the influence of anchor residues on peptide binding. The length of peptides (9- to 11-mer) did not affect A^*2402 binding except 8-mer peptides. Peptides possessing the aromatic residues at their C-terminus bound to A^*2402 molecules stronger than those bearing the aliphatic hydrophobic residues. These results indicate that two aromatic hydrophobic anchor residues permit the binding of longer peptides to A^*2402 molecules. Compared to our recent studies of B^*3501 and B^*5101 binding peptides, the present study suggested that both B and F pockets of A^*2402 molecules might be large and deep because these pockets favored bulky aromatic residues.     

Surface-expressed invariant chain (CD74) is required for internalization of human leucocyte antigen-DR molecules to early endosomal compartments

Transport of major histocompatibility complex (MHC) class II molecules to the endocytic route is directed by the associated invariant chain (Ii). In the endocytic pathway, Ii is proteolytically cleaved and, upon removal of residual Ii fragments, class II alpha beta dimers are charged with antigenic peptide and recognized by CD4+ T cells. Although distinct peptide-loading compartments such as MIIC (MHC class II loading compartment) and CIIV (MHC class II vesicles) have been characterized in different cells, there is growing evidence of a multitude of subcellular compartments in which antigenic peptide loading takes place. We employed a physiological cellular system in which surface Ii (CD74) and surface human leucocyte antigen (HLA)-DR were induced either alone or in combination. This was achieved by transient exposure of HT-29 cells to recombinant interferon-gamma (rIFN-gamma). Using distinct cellular variants, we showed that: (i) the majority of Ii molecules physically associate on the cell membrane with class II dimers to form DR alpha beta:Ii complexes; (ii) the presence of surface Ii is a prerequisite for the rapid uptake of HLA-DR-specific monoclonal antibodies into early endosomes because only the surface DR+/Ii+ phenotype, and not the DR+/Ii- variant, efficiently internalizes; and (iii) the HLA-DR:Ii complexes are targeted to early endosomes, as indicated by co-localization with the GTPase, Rab5, and endocytosed bovine serum albumin. Internalization of HLA-DR:Ii complexes, accommodation of peptides by DR alphabeta heterodimers in early endosomes and recycling to the cell surface may be a mechanism used to increase the peptide repertoire that antigen-presenting cells display to MHC class II-restricted T cells.     

The role of anchor residues in the binding of peptides to HLA-A*1101 molecules

Abstract: The binding of 136 8- to 12-mer peptides carrying anchor residues at position 2 (P2) and the C-terminus to HLA-A*1101 molecules was analyzed by a stabilization assay using RMA-S transfectants expressing HLA-A*1101 and human β₂-microglobulin. 72.1% of these peptides bound to HLA-A*1101 molecules. Two known HLA-All-restricted cytotoxic T-lymphocyte epitope peptides showed high affinity to HLA-A*1101. The results confirmed a previous pool sequencing study of HLA-A*1101 binding self-peptides, which showed that Lys at the C-terminus and Val, Ile, Phe, Tyr, and Thr at P2 are anchor residues for HLA-A*1101. Thr and aliphatic hydrophobic residues Val, Ile, and Leu at P2 are stronger anchor residues than the aromatic hydro-phobic residues Phe and Tyr. In addition, hydrophobic residues Leu, Phe, Tyr, Ile, and Ala at position 3 (P3) are secondary anchors but are weaker than those at P2. The affinities of the 8- and 12-mer peptides were significantly lower than those of 9- to 11-mer peptides. There was however no difference in affinity between 9-, 10- and 11-mer peptides. Furthermore, the analysis using peptides mutated at the C-terminus showed that HLA-A*1101 molecules can bind peptides carrying another positively charged residue, Arg. The present study clarified the role of the anchor residues at P2, P3 and the C-terminus in the binding of HLA-A*1101 molecules.     

Role of human immunodeficiency virus and cytomegalovirus in AIDS encephalitis.

Approximately half of patients with advanced acquired immune deficiency syndrome (AIDS) develop a subcortical dementia. The brains of all autopsies on AIDS patients performed at UCSD between 1982 and 1986 (N = 93) were studied. Neuropathologic changes consistent with a viral encephalitis were present in 54 brains (58%). Human immunodeficiency virus (HIV) antigens were detected in 37 of the brains (40%), most frequently in macrophages, multinucleated giant cells, and endothelial cells. Cytomegalovirus (CMV) was detected in 31 of the brains (33%), 22 of which also contained HIV. Cellular localization of CMV antigens suggests that CMV disseminates to the central nervous system hematogenously where the virus can infect endothelial cells, glia, and neurons. While the temporal course of the appearance of these two viruses within the CNS is not clear, the common simultaneous occurrence of both viruses within the brains of AIDS patients suggests that in vivo interaction between them may play a role in the pathogenesis of AIDS-associated encephalitis. Given the significant neurologic symptoms described in AIDS patients, the paucity of viral antigens suggests a pathogenic mechanism of indirect CNS damage rather than direct viral infection.     

Role of anchor residues in peptide binding to three HLA-A26 molecules

To investigate the role of anchor residues in HLA-A26 binding peptides, we analyzed the binding of various peptides to three HLA-A26 molecules using the HLA class I stabilization assay. Of twenty nonamer peptides carrying anchors at P2 and P9, 3, 6 and 3 peptides bound to HLA-A*2601, HLA-A*2602 and HLA-A*2603, respectively The peptide EV-IPMFSAL bound most strongly to these three HLA-A26 molecules. Analysis using mutants of this peptide at P1, P2 or P9 showed that acidic amino acids at P1 and five hydrophobic residues (Val, Thr, Ile, Leu and Phe) at P2 are anchor residues for the three HLA-A26 molecules while with exception of positively charged amino acids, a broad range of amino acids function as P9 anchor residues. These anchors were further evaluated using 38 nonamer peptides carrying anchor residues at P1, P2 and P9. Nineteen of these peptides bound to at least one HLA-A26 molecule. The frequency of HLA-A26 binding peptides was higher for peptides carrying all three anchor residues than for peptides carrying only P2 and P9 anchor residues. These results indicate that in addition to P2 and P9 anchors, the P1 anchor plays an important role in peptide binding to three HLA-A26 molecules.     

Role of human immunodeficiency virus infection in the pathogenesis of human papillomavirus-associated cervical neoplasia.

Although many basic questions about the relationship between HIV and HPV infection remain unresolved, epidemiological studies have consistently shown a strong association between HIV infection and the development of HPV-related squamous intraepithelial neoplasia. This work indicates that HIV infection may promote the clinical manifestation of subclinical or latent HPV infection. Recent technical advances localizing virus DNA and gene products in situ will provide new avenues for investigation, allowing us to go beyond correlations and to clarify the mechanisms of interaction between the two viruses in individual patients. With improved antiretroviral therapy and prophylaxis for HIV-associated opportunistic infection and prolonged survival of women with HIV, HPV infection and its most serious consequence, cervical cancer, are likely to assume greater significance in the clinical management of HIV-infected women throughout the world. A better understanding of the role of HIV in promoting the clinical manifestation of HPV infection will be essential to the control of this disease.     

Role of dendritic cells in immunopathogenesis of human immunodeficiency virus infection.

The role of dendritic cells (DC) in the pathogenesis of human immunodeficiency virus (HIV) disease has been a subject of considerable interest for several years. Initial studies focused on the infection, dysfunction, and depletion of DC in HIV-infected individuals. More recent studies have begun to identify the functional role of DC in the initiation and propagation of viral replication in T cells in HIV-infected individuals. This review discusses recent data regarding the role of DC in HIV disease with the aim of delineating basic immunopathogenic principles of infection and the development of therapeutic strategies.     

T-helper reactivity to simian immunodeficiency virus gag synthetic peptides in human immunodeficiency virus type 2 infected individuals

West African populations are infected with divergent strains of human immunodeficiency virus type 2 (HIV21, some of which are closely related to simian immunodeficiency virus (SIV) and it has been postulated that the HIV2 epidemic might have arisen by cross-species spread of SIV into the human population in West Africa. To gain some insight into the possible basis for cross protection between these two closely related viruses, the T-helper responses to 15 synthetic peptides from SIV gag synthetic peptides were investigated in seven HIV2-infected subjects and in seven healthy controls. Significant reactivity to at least one of the synthetic peptides tested was found in all patients and a statistically significant correlation between CD4+ lymphocyte absolute numbers and the number of reacting peptides was observed. A marginal lymphocyte reactivity was found in two of the healthy controls studied. In conclusion, this preliminary evidence that HIV2-infected patients exhibit T-cell responses to SIV gag peptides suggests that both viruses share t-helper epitopes in the gag viral region and raises the possibility of cross protection between SIV and HIV2 which may be relevant for HIV2 vaccine research based on closely related retroviruses. © 1995 WiIey-Liss, Inc.     

Role of non-anchor residues of D-restricted peptides in class I binding and TCR triggering

To understand better, the role of non-anchor residues of class I restricted T cell epitopes in class I binding and TCR stimulation, a panel of peptides was synthesized in which each of the nonanchor positions of the D^b-restricted influenza peptide, ASNENMETM, was changed to each of the 20 natural amino acids (AAs). The relative affinity of all the peptides for D^b was determined and their ability to stimulate anti-ASNENMETM cytotoxic T cell hybridomas was also assessed. The results illustrated that for D^b binding, the AAs with the most solvent exposure had the smallest effect on binding. Changes at other positions affected binding to different degrees. Results for the recognition by the T cell hybridomas indicated that a peptide-MHC complex represents a multitude of epitopes, as each hybridoma recognized a different subset of peptides. Most changes in the highly solvent-exposed exposed residues negatively affected recognition by all hybridomas while changes in other positions affected each hybridoma differently, independent of the direction of the side chain of the AA at that position. Furthermore, the use of saturating concentrations of low and high binding peptides showed that, as long as the class I-peptide complex is formed, the T-cell receptor does not differentiate between high and low binding peptides. This indicates that, although the stability of the class I-peptide complex is highly dependent on peptide affinity, the class I MHC conformation induced by low affinity peptides does not necessarily differ significantly from that induced by high affinity peptides. The results of peptide-class I recognition by one ASNENMETM-specific hybridoma was used to construct a peptide that differed from ASNENMETM at four of the nine residues, yet stimulated the hybridoma to a level comparable to ASNENMETM. In addition, peptides bearing the canonical D^b-binding motif but unable to bind to the class I molecule with high affinity could be made to bind D^b, by changing unfavorable AAs to favorable ones at appropriate positions. The extended motif determined was used to identify more accurately the peptides derived from Coxsakie b3 virus that would bind D^b. It was also shown that some of the canonical characteristics of the peptide motif could be obviated and still obtain high affinity binding, provided optimal AAs were present at secondary anchor positions.     

Acute human immunodeficiency virus infection.

Human immunodeficiency virus type 1 (HIV-1) infection has a broad spectrum of clinical manifestations, ranging from asymptomatic seroconversion to a severe symptomatic illness resembling infectious mononucleosis or other medical conditions including hepatitis, meningoencephalitis, or pneumonitis. Without clinical alertness, the illness is usually misdiagnosed or even not considered. Here we report 3 cases of acute HIV-1 infection with either a negative HIV-1 antibody assay or an indeterminate Western blot result, but high plasma levels of HIV-1 RNA. The initial presentations included fever, skin rash, sore throat, neck lymphadenopathy, cough and headache. One patient presented with infectious mononucleosis-like illness, 1 with aseptic meningitis, and 1 with acute tonsillitis. Physicians should be alert to the possibility of acute HIV-1 infection, especially in cases with unexplained fever, lymphadenopathy or rash.     

Pediatric human immunodeficiency virus infection.

In the past decade, an increase in pediatric human immunodeficiency virus (HIV) infection has had a substantial impact on childhood morbidity and mortality worldwide. The vertical transmission of HIV from mother to infant accounts for the vast majority of these cases. Identification of HIV-infected pregnant women needs to be impoved so that appropriate therapy can be initiated for both mothers and infants. While recent data demonstrate a dramatic decrease in HIV transmission from a subset of women treated with zidovudine during pregnancy, further efforts at reducing transmission are desperately needed. This review focuses on vertically transmitted HIV infection in children, its epidemiology, diagnostic criteria, natural history, and clinical manifestations including infectious and noninfectious complications. An overview of the complex medical management of these children ensues, including the use of antiretroviral therapy. Opportunistic infection prophylaxis is reviewed, along with the important role of other supportive therapies.     

Role of asparagine-linked glycosylation in human immunodeficiency virus type 1 transmembrane envelope function.

Transmembrane envelope protein (TM) residues 100, 105, and 128 of human immunodeficiency virus type 1 (HIV-1) strain HXB2 are potential sites for asparagine-linked oligosaccharide additions which are conserved among HIV-1 isolates, and all other lentivirus TM proteins. Site-specific mutants of each of the asparagine residues did not eliminate the ability of the virus to infect and replicate in CD4+ cells, but infectivity was reduced with all of these mutants, and syncytia induction was attenuated with two of these mutants. Studies of envelope expression of the mutant with the most severe defect demonstrated no significant effects on envelope protein synthesis, conformation, processing, multimerization, or release into the culture medium, suggesting that N-linked oligosaccharides are important in the specific fusion activity of TM.     

Towards a better definition of human leucocyte surface molecules

At the 4th International Workshop on Human Leucocyte Differentiation Antigens workers from 525 laboratories jointly evaluated and/or submitted about 1100 different monoclonal antibodies to human leucocyte surface molecules. At the final conference of the workshop agreement was reached on 35 new cluster determinants (CDs) and subclusters. In addition, seven previously established clusters were redefined. This report provides basic information concerning the molecules defined by these antibodies and some highlights of the conference.     

Conformation of human leucocyte antigen-C molecules at the surface of human trophoblast cells

Human leucocyte antigen (HLA)-C is expressed at lower levels than other classical HLA-I molecules on somatic cells. Surface HLA-C proteins can occur as conventionally beta(2)-microglobulin (beta2m)-associated complexes or as open conformers dissociated from peptide and/or beta(2)m. We investigated the conformation of HLA-C molecules on normal human trophoblast cells, which invade the maternal decidua during placentation. A panel of monoclonal antibodies to different conformations of HLA-I molecules was used in flow cytometry and surface immunoprecipitation experiments. On the surface of trophoblast cells only beta(2)m-associated complexes of HLA-C molecules were detected. In contrast, both open conformers and beta(2)m-associated HLA-C could be detected on other cells from the decidua, HLA-C-transfectants and cell lines. The levels of HLA-C expressed on primary trophoblast cells could be detected by antibodies specific to non-beta(2)m-associated conformations because binding was seen after acid-induced denaturation of surface proteins. In contrast to HLA-G molecules on trophoblasts, we found no evidence for the presence of disulphide-linked multimers of HLA-C complexes. These results show that most HLA-C molecules present at the trophoblast cell surface are in the conventional beta(2)m-associated conformation. These findings have implications regarding the stability of trophoblast HLA-C molecules and how they interact with receptors on decidual leucocytes during placentation.     

Inhibition of human immunodeficiency virus infection in monocytes by monoclonal antibodies against leukocyte adhesion molecules.

CD4 is the surface receptor for HIV envelope. Some evidence exists, however, that other cell surface receptors may be involved in viral entry subsequent to the initial binding of gp120 to CD4. Antibodies to leukocyte integrin LFA-1, a major component of intercellular adhesive interactions, have been shown to inhibit HIV-induced syncytia formation. Using a stringent system for in vitro HIV infection of human leukocytes, we examine the ability of some monoclonal antibodies (mAb) against various adhesion-related molecules to block or partially inhibit productive viral replication. HIV-1 infection of target monocytes or T cells by cell-free virus was blocked completely or partially by some mAb that prevent cell-cell interactions (CD4, HLA-DR, LFA-1, LFA-3), but not by others (ICAM-1, MAC-1, gp150.95, CD2, CD3, CD14). The capacity for mAb to block HIV infection appears to be epitope-specific, and does not relate to the ability to block homotypic adhesion. HIV transmission from infected cells was more difficult to block than was infection by cell-free virus. Adhesion molecules may be involved in facilitating early stages of HIV infection, following gp120/CD4 binding but prior to viral integration, in a manner distinct from cell-cell adhesion.     

Role of human immunodeficiency virus in leukocytes apoptosis from infected patients

The hallmark of the immunodeficiency virus infection is a progressive detriment of the immune response which has been associated to a gradual loss of its responsible components, in particularly, CD4 positive T cells. Although this cell population is considered the main target of the virus, there is a recent deal of interest in studying other components that may not be targets of the virus, but are important elements to control infectious microorganisms and that have been demonstrated to be altered during HIV infection. Neutrophils (PMN) are innate immune components that play a fundamental role against HIV infection and these cells have been described as functionally altered during AIDS. It has been suggested that such a dysfunction could be attributed to an increased susceptibility of these cells to accelerated spontaneous apoptosis. However, the underlying mechanisms that induce programmed cell death of neutrophils remain unknown. In previous works we have explored some events involved during cell death of neutrophils from HIV infected patients. It is the purpose of this work to review the current knowledge of apoptosis signals in neutrophils and to discuss our own data about some mechanisms involved in spontaneous and Fas mediated apoptosis, which may contribute to understand neutrophils dysfunction during HIV infection.     

Amino acid residues of the human immunodeficiency virus type I gp120 critical for the binding of rat and human neutralizing antibodies that block the gp120-sCD4 interaction.

We have characterized the discontinuous epitopes recognized by two rat and three human neutralizing monoclonal antibodies (mAb) by examining the effect of single amino acid changes in conserved residues of gp120 on mAb recognition. A human mAb derived from an infected individual, 448D, and two rat mAbs, 39.13g and 39.3b, respectively, derived by immunization with native recombinant gp120, recognize similar epitopes. Recognition of the envelope glycoproteins by these mAbs was affected by changes in gp120 amino acid residues 88, 113, 117, 257, 368, or 370. The gp120 amino acids 257, 368, and 370 have previously been reported to be important for CD4 binding, which is consistent with the ability of these mAbs to block the gp120-CD4 interaction. Residues 88, 113, and 117 are not thought to be important for CD4 binding, suggesting that the antibody epitopes overlap, but are distinct from, the CD4 binding region. We also found that some alterations in gp120 residues 88, 117, 368, or 421 reduced the ability of polyclonal sera from HIV-1-infected individuals to inhibit the interaction of the mutant gp120 glycoproteins with soluble CD4. Thus, changes in the HIV-1 gp120 glycoprotein that minimally affect the receptor binding may allow escape from neutralizing antibodies directed against the CD4 binding region.