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H Lewicki

Publications and source records attributed to H Lewicki.

29 records · Page 2Linked to original sources
PubMed1992

Novel LCMV-specific H-2k restricted CTL clones recognize internal viral gene products and cause CNS disease.

H-2k (C3H/Hej) cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were cloned. Three clones recognizing internal viral antigens were studied. One such CTL clone recognized neither the glycoprotein nor nucleoprotein encoded by the viral short RNA segment, but reacted with a protein encoded by the long RNA segment, either the viral polymerase, or the Z protein. This one clone, in addition to primary CTL harvested from immunized C3H mice, failed to lyse target cells expressing the Z protein, suggesting recognition was to the viral polymerase. Two other clones recognized the viral nucleoprotein, amino acids 93-100, as determined by protein deletion and peptide mapping studies. When introduced directly into the central nervous systems of LCMV-infected histocompatible mice, all clones were active in vivo and capable of causing immunopathologically mediated death.

Animals↗
PubMed1992

Diversity of T-cell receptors in virus-specific cytotoxic T lymphocytes recognizing three distinct viral epitopes restricted by a single major histocompatibility complex molecule.

Cytotoxic T lymphocytes (CTL) recognize virus peptide fragments complexed with class I major histocompatibility complex (MHC) molecules on the surface of virus-infected cells. Recognition is mediated by a membrane-bound T-cell receptor (TCR) composed of alpha and beta chains. Studies of the CTL response to lymphocytic choriomeningitis virus (LCMV) in H-2b mice have revealed that three distinct viral epitopes are recognized by CTL of the H-2b haplotype and that all of the three epitopes are restricted by the Db MHC molecule. The immunodominant Db-restricted CTL epitope, located at LCMV glycoprotein amino acids 278 to 286, was earlier noted to be recognized by TCRs that consistently contained V alpha 4 segments but had heterogeneous V beta segments. Here we show that CTL clones recognizing the other two H-2Db-restricted epitopes, LCMV glycoprotein amino acids 34 to 40 and nucleoprotein amino acids 397 to 407 (defined in this study), utilize TCR alpha chains which do not belong to the V alpha 4 subfamily. Hence, usage of V alpha and V beta in the TCRs recognizing peptide fragments from one virus restricted by a single MHC molecule is not sufficiently homogeneous to allow manipulation of the anti-viral CTL response at the level of TCRs. The diversity of anti-viral CTL likely provides the host with a wider option for attacking virus-infected cells and prevents the emergence of virus escape mutants that might arise if TCRs specific for the virus were homogeneous.

Amino Acid Sequence↗
PubMed1991

Virus infection triggers insulin-dependent diabetes mellitus in a transgenic model: role of anti-self (virus) immune response.

We investigated the potential association between viruses and insulin-dependent (type 1) diabetes (IDDM) by developing a transgenic mouse model. By inserting into these mice a unique viral protein that was then expressed as a self-antigen in the pancreatic islets of Langerhans, we could study the effect on that expressed antigen alone, or in concert with an induced antiviral (i.e., autoimmune) response manifested later in life in causing IDDM. Our results indicate that a viral gene introduced as early as an animal's egg stage, incorporated into the germline, and expressed in islet cells does not produce tolerance when the host is exposed to the same virus later in life. We observed that the induced anti-self (viral) CTL response leads to selective and progressive damage of beta cells, resulting in IDDM.

Animals↗
PubMed1991

Mapping the anatomy of the immunodominant domain of the human immunodeficiency virus gp41 transmembrane protein: peptide conformation analysis using monoclonal antibodies and proton nuclear magnetic resonance spectroscopy.

Thirty-six monoclonal antibodies from mice and three from rats were raised against a peptide corresponding to the immunodominant domain of the transmembrane gp41 protein of human immunodeficiency virus (HIV) type 1 (LGLWGCSGKLIC; amino acid residues 598 to 609). Of these, three monoclonal antibodies from the mice and one from a rat also reacted with the corresponding peptide derived from the HIV type 2 transmembrane gp41 protein (amino acid residues 593 to 603; NSWGCAFRQVC). Immunochemical studies using a variety of synthetic peptides indicated that the cross-reactivity was due to antibody binding to CSGKLIC of HIV type 1 or CAFRQVC of HIV type 2. Single amino acid substitutions for a cysteine at either the amino or the carboxy end of the peptide interrupted antibody binding, indicating that the site recognized was the Cys-XXXXX-Cys loop. Similar results were obtained when the 11-mer HIV type 2 gp41 peptide (amino acids 593 to 603) was inoculated into mice to raise monoclonal antibodies. In this instance, of 30 monoclonal antibodies developed, 4 reacted with both HIV type 1 and HIV type 2 peptides. The conformation of a seven-residue peptide, CSGKLIC, corresponding to residues 603 to 609 of the gp41 immunodominant epitope of HIV-1 was investigated by proton nuclear magnetic resonance spectroscopy. The immunologically active form of CSGKLIC contains an intramolecular disulfide bond and maintains a preference for a folded conformation, apparently including a type I reverse turn about the residues SGKL. No such preference is observed for the reduced form of the peptide, which contains two thiol groups. The presence of the disulfide bond is thus integral to the formation of the structure of the loop in solution. In agreement with this finding, elimination of the possibility of loop formation by substitution of S for C at the amino or carboxy termini of the 7-mer is accompanied by the failure of antibody binding to this peptide.

Amino Acid Sequence↗
PubMed1990

Cytotoxic T lymphocytes do not control lymphocytic choriomeningitis virus infection of BB diabetes-prone rats.

BioBreeding Worcester diabetes-prone (BBdp) rats develop insulin-dependent autoimmune-driven diabetes mellitus spontaneously and intravenous administration of 1 x 10(7) p.f.u. of lymphocytic choriomeningitis virus (LCMV) to young adult mice prevents disease. The virus is lymphotropic, binding to and replicating in such cells. BBdp rats fail to generate virus-specific major histocompatibility complex-restricted cytotoxic T lymphocyte (CTL) responses when challenged with this dose or other doses of LCMV, Pichinde virus or vaccinia virus. Yet such rats clear virus effectively and show no evidence of persistent infection. Associated with this clearance of virus and establishment of immunity is the production of neutralizing antibodies. In contrast, diabetes-resistant (BBdr) rats generate virus-specific CTL responses. Furthermore LCMV binds to fewer lymphoid cells of BBdr rats (in comparison to those of BBdp rats) and replicates in fewer lymphocytes (by one order of magnitude) from these rats. Thus, unlike mice in which CTLs play a dominant role in the control of LCMV infection, BBdp rats do not overcome this infection via CTLs. In addition, both the BBdp rats and their BBdr counterpart may provide useful models for determining whether or how individual lymphocyte subsets function in the induction of CTL responses, for the analysis of virus-induced immunosuppression and for the use of viruses or their products as therapeutic modalities.

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PubMed1989

Molecular analyses of a five-amino-acid cytotoxic T-lymphocyte (CTL) epitope: an immunodominant region which induces nonreciprocal CTL cross-reactivity.

The cytotoxic T-lymphocyte (CTL) response to lymphocytic choriomeningitis virus infection determines the outcome of infection. Here we show that this response in BALB/c mice (H-2d), when analyzed both at the primary CTL level and using CTL clones, is predominantly monospecific. The vast majority of CTL have a common specificity for a single epitope in the virus nucleoprotein, which can be minimally identified by amino acids GVYMG. This epitope is presented by the Ld class I glycoprotein. We used these data to design a subunit CTL vaccine, whose effectiveness is demonstrated in the accompanying report (L. S. Klavinskis, J. L. Whitton, and M. B. A. Oldstone, J. Virol. 63:4311-4316, 1989). Further analysis indicates that, while CTL clones share a common minimal epitope, they differ in their ability to recognize cells infected with a related but distinct strain of lymphocytic choriomeningitis virus. Studies on the molecular nature of CTL cross-reactivity indicate that CTL induced by similar sequences may cross-react in a unidirectional manner. These novel observations suggest that CTL vaccines, to achieve optimal effectiveness, should not simply include virus sequences which will yield a CTL response; the immunizing sequences should also be selected to ensure that the fine specificities of the induced CTL are such that they maximize the chance of recognizing serotypically diverse strains.

Amino Acid Sequence↗
PubMed1988

Fine dissection of a nine amino acid glycoprotein epitope, a major determinant recognized by lymphocytic choriomeningitis virus-specific class I-restricted H-2Db cytotoxic T lymphocytes.

We define a nine-amino acid (aa) sequence of VAL-GLU-ASN-PRO-GLY-GLY-TYR-CYS-LEU as a major epitope for immunologic recognition of lymphocytic choriomeningitis virus (LCMV) by H-2b-restricted CTL. The epitope was characterized using molecular genetics to bracket broadly and chemistry to precisely identify aa residues 278-286 of the viral glycoprotein. The epitope's composition is characteristic of a reverse (beta turn) but not an amphipathic alpha helix. A series of peptides with a single aa substitution in position 278 of VAL with other nonpolar (hydrophobic) amino acids (LEU, ILE, ALA, or GLY) coat targets that are recognized and lysed by CTL clones recognizing this epitope. In contrast, substitution of VAL with either large aromatic amino acids (that add bulk: PHE, TYR) or polar side chains (SER, THR) segregates CTL clones normally recognizing aa 278-286 into two groups, one that remains lytic (permissive) despite these changes and another that fails to lyse, indicating CTL can discriminate at a single aa. A change in charge at this position (VAL----ASP or GLU), in general, reduces CTL lysis while a change of VAL to LYS or ASN has minimal affect for four of the five CTL clones analyzed. CTL reactivity with the viral epitope is restricted by the Db but not the Kb of the murine MHC haplotype. A 16-aa peptide of Db that spans alpha 1 residues 37-52 blocks CTL lysis, whereas the corresponding Kb peptide that differs from Db in a single aa in position 50 does not.

Amino Acid Sequence↗
PubMed1988

Virus-lymphocyte interactions. III. Biologic parameters of a virus variant that fails to generate CTL and establishes persistent infection in immunocompetent hosts.

Viruses that cause in vivo persistent infections avoid the host's immunologic surveillance machinery. A major component of that armamentarium is virus-specific MHC-restricted cytotoxic T lymphocyte (CTL) response of the host. Studies with lymphocytic choriomeningitis virus (LCMV) have uncovered a parental virus (CTL+) that in immuno-competent adults induces CTL and terminates acute infection and a variant (CTL-) that fails to elicit CTL responses and establishes a persistent state (R. Ahmed et al. (1984) J. Exp. Med. 160, 521-540). The biologic properties, similarities, and differences between CTL+ and CTL- viruses as regards their interactions with lymphocytes of newborn and adult mice is recorded here. CTL+ and CTL- viruses persist in lymphocytes of newborn inoculated mice, primarily within the T helper subset. Approximately 2% of lymphocytes express viral nucleic acid sequences while only 0.04% score as infectious centers suggesting incomplete viral replication. These levels were maintained over the course of infectious. In contrast, CTL- virus but not CTL+ persists in lymphocytes of mice inoculated when adults. Lymphocytes easily scored as infecting centers but rarely displayed nucleic acid sequences suggesting a different balance of incomplete to complete virion replication. Further, infectious centers decreased by 10-fold from the 3rd to 68th day of infection and the total numbers of T lymphocytes in the circulation decreased suggesting CTL- may replicate in and destroy lymphocytes of adult mice. In the following paper the primary nucleotide structure of the LCMV small RNA segment, the segment responsible for generation of CTL and encoding the proteins recognized by CTL, for CTL+ and CTL- viruses is reported.

Animals↗
PubMed1988

Molecular definition of a major cytotoxic T-lymphocyte epitope in the glycoprotein of lymphocytic choriomeningitis virus.

Analyses with segmental reassortants of lymphocytic choriomeningitis virus (LCMV) RNA have shown that cytotoxic T lymphocytes (CTL) are induced by and recognize proteins encoded by the viral short segment, which specifies two virus structural proteins, glycoprotein (GP) and nucleoprotein (NP). Expression of cDNA copies of these genes in vaccinia virus vectors demonstrates that C57BL/6 (H2bb) mice mount significant CTL responses to both GP and NP. We have used LCMV-specific H2bb-restricted CTL clones and a family of serial C-terminal truncations of the LCMV GP expressed in vaccinia virus to map the precise specificities of the anti-GP clones. Of the 18 CTL clones studied, 1 recognizes NP and the other 17 recognize GP. The reactivities of 14 of the 17 anti-GP CTL clones against the deleted GP molecules have been fully characterized, and two clear patterns of anti-GP activity have emerged, defining at least two CTL epitopes. The first epitope, recognized by only two of the clones, lies within GP residues 1 to 218. The second is recognized by all 12 of the remaining clones and was mapped, by using the GP deletions, to a 22-amino-acid region comprising GP residues 272 to 293. A synthetic peptide representing this area sensitized uninfected syngeneic target cells to lysis both by bulk CTL obtained from the spleen after a primary immunization and by appropriate CTL clones. Two sets of criteria are available which are said to identify potential T-cell epitopes, one based on primary amino acid sequence and the second based on protein secondary structure. Neither of these predictive schemes would have identified region 272 to 293 as a CTL recognition motif, indicating that such programs are of limited usefulness as presently conceived. Analysis of the CTL clones shows clearly that all three families (anti-NP and anti-GP 1 to 218 and 272 to 293) direct efficient cross-reactive killing against a variety of serologically distinct strains of LCMV.

Animals↗
PubMed1985

Properties and characterization of monoclonal antibodies to Tacaribe virus.

Monoclonal antibodies prepared against Tacaribe and Junin viruses have been used to define further the serological relationships between arenaviruses of the Tacaribe complex. A close relationship was found between these two viruses and the heterologous Amapari and Machupo viruses, with Pichinde virus and Parana virus being more distantly related. Among the antibodies specific for Tacaribe virus, five were found to react with viral antigens at the surface of infected cells and to neutralize virus infectivity in vitro. These five antibodies could be differentiated by competitive immunoassay as recognizing at least two antigenically distinct epitopes. The kinetics of reaction between antibody and virus were examined for all five neutralizing antibodies. One antibody (2.25.4) effectively neutralized all infectious virus. The remaining four directed against a second epitope gave significant persistent fractions which could be reduced by addition of complement, anti-mouse immunoglobulin, or antibody 2.25.4. Variants of Tacaribe virus resistant to neutralization by antibody 2.25.4 were obtained by growth in the presence of this antibody and neutralization kinetics were reexamined using the heterologous monoclonal neutralizing antibodies. Several different neutralization profiles were obtained, suggesting that point mutations resulted in conformational changes at topographically selected distinct epitopes recognized by the remaining antibodies.

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