The minimum peptide epitope from the influenza virus matrix protein. Extra and intracellular loading of HLA-A2.

Influenza virus matrix protein-derived peptides were synthesized based on the amino acid motifs for HLA-A2 bound self peptides. Among these peptides a nonamer (amino acids 58 through 66: G I L G F V F T L) was found to be 100 to 1000 times more effective than the commonly used peptide 57-68 (K G I L G F V F T L T V) in sensitizing HLA-A2+ target cells to lysis by influenza virus specific cytotoxic T lymphocytes. The sensitizing activity of the 12-mer 57-68 was not due to contamination with shorter and more active peptides. Intracellular expression of peptide 58-66 (mediated by a stable expression plasmid with DNA coding for this peptide) also sensitized HLA-A2+ cells to lysis. Peptide 58-66 stimulated human PBMC to generate CTL that recognized peptides 58-66 and 57-68 in association with HLA-A2.     

Specificity of peptide binding by the HLA-A2.1 molecule

The HLA-A2 molecule contains a putative peptide binding site that is bounded by two alpha-helices and a beta-pleated sheet floor. Previous studies have demonstrated that the influenza virus matrix peptide M1 55-73 can sensitize target cells for lysis by HLA-A2.1-restricted virus-immune CTL and can induce CTL that can lyse virus-infected target cells. To assess the specificity of peptide binding by the HLA-A2.1 molecule, we examined the ability of seven variant M1 peptides to be recognized by a panel of M1 55-73 peptide-specific HLA-A2.1-restricted CTL lines. The results demonstrate that five out of the seven variant M1 55-73 peptides could be recognized by A2.1-restricted M1 55-73 peptide-specific CTL lines. The two variant peptides that were not recognized by any CTL could bind to HLA-A2.1 as indicated by their ability to compete for presentation of the M1 55-73 peptide. In addition, 5 of a panel of 24 unrelated peptides tested could also compete for M1 55-73 presentation by HLA-A2.1. One peptide derived from the sequence of a rotavirus protein could sensitize HLA-A2.1+ targets for lysis by M1 55-73 peptide-specific CTL. We conclude from these studies that: 1) the HLA-A2.1 molecule can bind a broad spectrum of peptides; 2) T cells selected for the ability to recognize one peptide plus a class I molecule can actually recognize an unrelated peptide presented by that same class I molecule; and 3) a stretch of three adjacent hydrophobic amino acids may be an important common feature of peptides that can bind to HLA-A2.1.     

Comparison between two peptide epitopes presented to cytotoxic T lymphocytes by HLA-A2. Evidence for discrete locations within HLA-A2

An influenza B virus nucleoprotein (BNP) peptide, residues 82-94, defined by limited sequence homology with an HLA-A2-restricted peptide from influenza A matrix protein, was recognized by HLA-A2-restricted CTL. Reciprocal inhibition of T cell recognition by the two peptides suggest that the BNP peptide may have lower avidity for HLA-A2 molecules than the matrix peptide. The interaction between this peptide and HLA-A2 was explored by studying the CTL recognition of BNP 82-94 presented by mutant HLA-A2 molecules. Mutations at residues 9, 99, 70, 74, 152 and 156 were found to abolish T cell recognition of the BNP peptide. These results were compared with results previously obtained with the influenza A matrix peptide and suggest that the two peptides bind differently in the peptide binding site.     

The 45 pocket of HLA-A2.1 plays a role in presentation of influenza virus matrix peptide and alloantigens

Amino acid substitutions were introduced into the 45 pocket of HLA-A2.1 to determine the potential role of this structurally defined feature of class I molecules in viral peptide and alloantigen presentation. The 45 pocket lies below the alpha 1-domain alpha-helix and is composed of five amino acids, three of which differ between HLA-A2.1 and HLA-B37. These two class I molecules have previously been shown to have largely non-overlapping peptide-binding specificities. Site-directed mutagenesis was used to replace the hydrophobic residues at positions 24, 45, and 67 in the 45 pocket of HLA-A2.1 with the hydrophilic amino acids found in these positions in HLA-B37. Thus, three single amino acid mutants were produced: 24A----S, 45 M----T, and 67V----S. These mutants were transfected into HMy2.C1R cells and assessed for their ability to present influenza virus matrix M1 57-68 peptide and HTLV-I Tax-1 2-25 peptide to HLA-A2.1-restricted, peptide-specific CTL and to present alloantigens to HLA-A2-allospecific CTL lines. Each of these substitutions in the 45 pocket produced a molecule that failed to present the M1 peptide to most M1 peptide-specific CTL lines. In contrast, none of these mutations affected presentation of the Tax-1 peptide to Tax-1-specific CTL lines, which indicates that these mutant HLA-A2 molecules can function in viral peptide presentation. Two of the three substitutions in the 45 pocket resulted in lack of recognition by a subset of HLA-A2 allospecific CTL lines. These results demonstrate that the amino acid side chains in the 45 pocket can strongly influence peptide presentation and suggest that the 45 pocket may play a role in determining peptide-binding specificity.     

Species specificity in the interaction of CD8 with the alpha 3 domain of MHC class I molecules.

The alpha 1 and alpha 2 domains of the class I MHC molecule constitute the putative binding site for processed peptides and the TCR, although the alpha 3 domain has been implicated as a binding site for the CD8 molecule. Species specificity in the binding of CD8 to the alpha 3 domain has been suggested as an explanation for the low xenogeneic T cell response to class I molecules, but results on this point have been conflicting and controversial. We have addressed this issue using CTL lines from HLA-A2.1 transgenic mice that specifically recognize and lyse A2.1-expressing cells infected with influenza A/PR/8 or pulsed with influenza matrix peptide M1(57-68). Species specificity was examined using transfectants that expressed hybrid molecules containing the alpha 1 and alpha 2 domains from HLA-A2.1 and the alpha 3 domain from a murine class I molecule. Lower levels of M1(57-68) peptide were required to sensitize L cell transfectants expressing a chimera that contained an H-2Dd alpha 3 domain than targets expressing the intact A2.1 molecule. However, at high doses of peptide, lysis of these two targets was similar. However, no reproducible difference in sensitization was observed using EL4 or Jurkat transfectants expressing A2.1 or A2.1 chimeric molecules that contained an H-2Kb alpha 3 domain. In all cases, however, lysis of peptide-pulsed A2.1 expressing targets was more sensitive to inhibition with anti-CD8 mAb than lysis of cells expressing these chimeric molecules. Thus, under suboptimal conditions such as low Ag density or in the presence of anti-CD8 mAb, these CTL preferentially recognize class I molecules with a murine alpha 3 domain. This suggests that there is some species specificity in the interaction of CD8 with the alpha 3 domain of the class I molecule. However, CTL recognition was inhibited by point mutations in the alpha 3 domain of HLA-A2.1 that have been shown to inhibit binding of human CD8 and recognition by human CTL, suggesting that murine CD8 interacts to some degree with human alpha 3 domains, and that similar alpha 3 domain residues may be important for murine and human CD8 binding. The relevance of these results to an understanding of low xenogeneic responses is discussed.     

Two epitopes and one agretope map to a single HLA-A2 peptide recognized by H-2-restricted T cells

Mice immunized with syngeneic cells transfected with cloned genes coding for HLA class I molecules could recognize the human MHC Ag in the context of their own H-2 molecules. We obtained CTL clones from DBA/2 mice (H-2d) which had been immunized with P815 cells (a mastocytoma of DBA/2 origin) expressing either HLA-A2 or HLA-A3 or two different molecules containing recombined sequences of HLA-A2 and HLA-A3. Fourteen of these clones recognized a synthetic peptide corresponding to the region 170-185 of HLA-A2 in the context of H-2Kd. Moreover, from their activity on P815 cells expressing HLA-Cw3, two subpatterns could be distinguished: subpattern Cw3+, defined by those clones which lysed P815-Cw3, and subpattern Cw3- defined by those clones which did not lyse P815-Cw3. By testing the activity of clones of each subpattern on a series of modified synthetic peptides, we were able to define two epitopes on the same 170-185 peptide of HLA-A2. One of them was dependent on amino acids at positions 173 and 177, whereas the other was dependent on amino acid 177 alone. By using competition experiments, we were also able to define an agretopic region strongly dependent on the amino acid at position 178. Furthermore, experiments with L cells expressing molecules containing recombined sequences between H-2Kd and H-2Dd demonstrated the determinant role of residues 152, 155, and 156 from H-2Kd in the presentation to murine T cells of the 170-185 peptide of HLA-A2.     

Identification of natural antigenic peptides of a human gastric signet ring cell carcinoma recognized by HLA-A31-restricted cytotoxic T lymphocytes.

Peptides of human melanomas recognized by CD8+ CTLs have been identified, but the nature of those of nonmelanoma tumors remains to be elucidated. Previously, we established a gastric signet ring cell carcinoma HST-2 and HLA-A31 (A*31012)-restricted autologous CTL clone, TcHST-2. In the present study, we determined the natural antigenic peptides of HST-2 cells. The purified preparation of acid-extracted Ags was submitted to the peptide sequencer, and one peptide, designated F4.2 (Tyr-Ser-Trp-Met-Asp-Ile-Ser-Cys-Trp-Ile), appeared to be immunogenic. To confirm the antigenicity of F4.2 further, we constructed an expression minigene vector (pF4.2ss) coding adenovirus E3, a 19-kDa protein signal sequence plus F4.2. An introduction of pF4.2ss minigene to HST-2 and HLA-A31(+) allogeneic tumor cells clearly enhanced and induced the TcHST-2 reactivity, respectively. Furthermore, when synthetic peptides of F4.2 C-terminal-deleted peptides were pulsed to HST-2 cells, F4.2-9 (nonamers), but not F4.2-8 or F4.2-7 (octamer or heptamer, respectively), enhanced the reactivity of TcHST-2, suggesting that the N-terminal ninth Trp might be a T cell epitope. This was confirmed by lack of antigenicity when using synthetic substituted peptides as well as minigenes coding F4.2 variant peptides with Ala or Arg at the ninth position of F4.2. Meanwhile, it was indicated that the sixth position Ile was critically important for the binding to HLA-A31 molecules. Thus, our data indicate that F4.2 may work as an HLA-A31-restricted natural antigenic peptide recognized by CTLs.     

A single amino acid substitution in HLA-A2 can alter the selection of the cytotoxic T lymphocyte repertoire that responds to influenza virus matrix peptide 55-73

Previous studies have demonstrated that certain amino acid substitutions in the alpha two domain at positions 152 and 156 in the alpha two helix of the HLA-A2 molecule can affect presentation of the influenza virus matrix peptide M1 55-73 without abolishing binding of the M1 peptide. HLA-A2.1-restricted M1 55-73 peptide-specific CTL lines obtained from almost all HLA-A2.1+ individuals fail to recognize the M1 peptide presented by site-directed mutants of HLA-A2 that have either a Val----Ala or Val----Gln substitution at position 152 or a Leu----Trp substitution at position 156. Only one HLA-A2+ individual (donor Q66, HLA-A2,-B53,-B63) has been found who is able to generate a unique repertoire of HLA-A2-restricted M1 peptide-specific CTL that can recognize peptide presented by HLA-A2 mutants with either an Ala or Gln substitution at position 152 or a Trp substitution at position 156. These Q66 M1 peptide-specific CTL could be selected by stimulation with M1 peptide-pulsed transfectants that express the mutant HLA-A2 gene with the Trp substitution at 156. To determine if the presence of the unique CTL repertoire could be attributed to a variant HLA-A2 molecule in Q66, sequences were determined from polymerase chain reaction-amplified segments of the HLA-A2 RNA. Two different HLA-A2 genes were found expressed in Q66 cells: one is identical to HLA-A2.1 and the other is identical to HLA-A2.2F (Gln----Arg at position 43, Val----Leu at position 95, and Leu----Trp at position 156). These results demonstrate that a different CTL repertoire specific for HLA-A2 plus the M1 55-73 peptide is generated in an individual that expresses both HLA-A2.1 and HLA-A2.2F compared to individuals who express HLA-A2.1 alone, and that the unique repertoire can be selected by the presence of an HLA-A2 molecule with a single amino acid substitution at position 156.     

Synthesis of peptide fragments of influenza virus A/Aichi/2/68 (H3N2) hemagglutinins

Peptides corresponding to sequences 122-133, 136-147, and 154-164 of the heavy chain of hemagglutinin of the A/Aichi/2/68 (H3N2) influenza virus have been synthesized by stepwise elongation of the peptide chain with Boc-amino acid activated esters or by condensation of peptide blocks by DCC/HOBt-method. A coloured C-protecting group, 2-[4-(phenylazo)-benzylsulfonyl]ethyl (PSE), was used, which is convenient in purification of synthetic peptides. After removal of terminal N-and C-protecting groups the side-protecting residues were cleaved off with 1 M trifluoromethanesulfonic acid in trifluoroacetic acid containing 10% thioanisole. Crude products were purified by preparative reversed-phase liquid chromatography. Synthesized peptides were conjugated with BSA.     

Sequence motifs important for peptide binding to the human MHC class I molecule, HLA-A2.

Previous studies have indicated that most HLA-A2-binding peptides are 9 amino acid (aa) residues long, with a Leu at position 2 (P2), and a Val or Leu at P9. We compared the binding properties of different peptides by measuring the rate of dissociation of beta 2-microglobulin from peptide-specific HLA-A2 complexes. The simplest peptide that we identified that could form HLA-A2 complexes had the sequence (in single letter aa code) GLFGGGGGV, indicating that three nonglycine aa are sufficient for binding to HLA-A2. To determine whether most nonapeptides that contained Leu at P2 and Val or Leu at P9 could bind to HLA-A2, we tested the binding of nonapeptides selected from published HIV and melanoma protein sequences, and found that six of seven tested formed stable HLA-A2 complexes. We identified an optimal antigenic undecapeptide from the cytomegalovirus gB protein that could form stable HLA-A2 complexes that contained apparent anchor residues at P2 and P11 (sequence FIAGN-SAYEYV), indicating that the spacing between anchor residues can be somewhat variable. Finally, we tested the importance of every aa in the influenza A matrix peptide 58-66 (sequence GILGFVFTL) for binding to HLA-A2, by using Ala-substituted and Lys-substituted peptides. We found that multiple positions were important for stable binding, including P2, P3, P5-P7, and P9. We conclude that the P2 and P9 anchor residues are of prime importance for peptide binding to HLA-A2. However, other peptide side chains (especially at P3) contribute to the stability of the interaction. In certain cases, the optimal length for peptide binding can be longer than 9 residues.     

Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge.

The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection.     

A CTL-based liposomal vaccine capable of inducing protection against heterosubtypic influenza viruses in HLA-A*0201 transgenic mice

The current vaccination strategy against influenza is to induce the production of antibodies directed against surface antigens of viruses. However, the frequent changes in the surface antigens of influenza viruses allow the viruses to avoid antibody-mediated immunity. On the other hand, it is known that cytotoxic T-lymphocyte (CTL) populations directed against internal antigens of influenza A virus are broadly cross-reactive to influenza virus subtypes. In the present study, liposomal conjugates with CTL epitope peptides derived from highly conserved internal antigens of influenza viruses were evaluated for their ability to protect against infection with influenza viruses. Liposomal conjugates with peptide M1 58-66, an HLA-A*0201-binding CTL epitope present within the amino-acid sequence of the M1 coding region, successfully induced antigen-specific CD8⁺ T-cells and CTLs in HLA-A*0201-transgenic mice. Moreover, after nasal infection with either the H1N1 or H3N2 virus, viral replication in the lung was significantly inhibited in the immunized mice. These protective activities lasted at least 6 months after the immunization. Thus, these results suggest that liposome-coupled CTL epitope peptides derived from highly conserved internal antigens of influenza viruses might be applicable to the development of vaccines that induce protection against infection with heterosubtypic influenza viruses.     

Characterization of the Antiviral Activity for Influenza Viruses M1 Zinc Finger Peptides

We sought to investigate the cellular uptake and antiviral activity for the M1 zinc finger peptides derived from influenza A and influenza B viruses in vitro. No cellular uptake was detected by fluorescent microscopy for the synthetic zinc finger peptides. When flanked to a cell permeable peptide Tp10, the zinc finger recombinant proteins were efficiently internalized by MDCK cells. However, no antiviral activity was detected against homologous or heterologous virus infections for the synthetic peptides or the Tp10-flanked recombinant proteins, regardless treated with or without Zn²⁺. Nevertheless, MDCK cell constitutively expressing the M1 zinc finger peptides in cell nuclei potently inhibited replication of homologous, but not heterologous influenza viruses. Adenoviral vector delivered M1 zinc finger peptides also exhibited potent antiviral activity against homologous viruses challenge. Transduction at 100 PFU dose of recombinant adenovirus efficiently protected 99% of the cells from 100 TCID₅₀ of different virus infections for both peptides. These results brought new insight to the antiviral researches against influenza virus infections.     

Identification of melanoma-specific peptide epitopes by HLA-A2.1-restricted cytotoxic T lymphocytes

HLA-A2.1-associated peptides, extracted from human melanoma cells, were used to study epitopes for melanoma-specific HLA-A2.1-restricted cytotoxic T lymphocytes （CTLs） by epitope reconstitution, active peptide sequence characterization and synthetic peptide verification. CTL were generated from tumor-involved nodes by in vitro stimulation, initially with autologous melanoma cells and subsequently with allogeneic HLA-A2.1 positive melanoma cells. The CTLs could lyse autologous and aUogeneic HLA-A2. 1 positive melanomas, but not HLA-A2.1 negative melanomas or HLA-A2.1 positive non-melanomas. The lysis of melanomas could be inhibited by anti-CD3, anti-HLA class I and anti-HLA-A2.1 monoclonal antibodies. HLA-A2.1 molecules were purified from detergent-solubilized human melanoma cells by immunoaffinity column chromatography and further fractionated by reversed phase high performance liquid chromatography. The fractions were assessed for their ability to reconstitute melanoma-specific epitopes with HLA-A2.1 positive antigen-processing mutant T2 cells. Three reconstitution peaks were observed in lactate dehydrogenase release assay. Mass spectrometry and ion-exchange high performance liquid chromatography analysis were used to identify peptide epitopes. Peptides with a mass-to-charge ratio of 948 usually consist of nine amino acid residues. The data from reconstitution experiments confirmed that the synthetic peptides contained epitopes and that the peptides associated with HLA-A2.1 and recognized by melanoma-specific CTL were present in these different melanoma cells. These peptides could be potentially exploited in novel peptide-based antitumor vaccines in immunotherapy for CTL.     

Overlapping epitopes that are recognized by CD8+ HLA class I-restricted and CD4+ class II-restricted cytotoxic T lymphocytes are contained within an influenza nucleoprotein peptide.

Viral epitopes that are recognized by both HLA class I-restricted and class II-restricted T cells have been defined for a type A influenza virus nucleoprotein (NP) peptide. CD8+ and CD4+ CTL lines have been generated against a synthetic peptide encompassing residues 335 to 349 of NP that are restricted by HLA-B37 and HLA-DQw5, respectively. Both of these CTL populations were capable of specifically lysing influenza A virus-infected targets, indicating that a naturally processed NP peptide(s) was being mimicked by the NP (335-349) peptide. Amino acid residues that are critical for recognition of this NP determinant in the context of HLA-B37 and HLA-DQw5 were investigated by the use of panels of truncated and alanine-substituted NP peptides. The results demonstrate that: 1) truncations in the amino- or carboxy-terminal ends differentially affect CD8+ and CD4+ CTL recognition; 2) the NP (335-349) sequence contains two octapeptide epitopes that share a core of six amino acid residues (NP 338-343); and 3) alanine substitutions at five of these residues abrogated recognition by at least one of the CD8+ and CD4+ CTL lines. Thus, these class I- and class II-restricted CTL lines recognize similar but distinct epitopes, and different structural features of the NP peptide are required for presentation by HLA-B37 and HLA-DQw5. Comparison of the amino acid sequences of the NP peptide presented by HLA-B37 and HLA-DQw5 with other peptides known to be presented by both class I and class II molecules revealed a common motif among these peptides.     

The recognition of a viral antigenic moiety by class I MHC-restricted cytolytic T lymphocytes is limited by the availability of the endogenously processed antigen.

The transmembrane hydrophobic domain of the type A influenza A/JAPAN/305/57 (H2N2) hemagglutinin (HA) contains an immunodominant site encompassing amino acids 523-545 (J523-545) recognized by class I MHC-restricted cytolytic T lymphocytes (CTL). Class I CTL of two fine specificity subsets map to this transmembrane (TM) site. One of these CTL subpopulations is subtype specific. These T lymphocytes recognize the site generated during infection of target cells with A/JAPAN/305/57 virus (H2N2) but not target cells expressing the comparable TM site of the influenza A/PR/8/34 virus (H1N1) hemagglutinin (P527-549) after infection with this virus. The other CTL subpopulation is cross-reactive and recognizes the TM site of the A/JAPAN/305/57 HA and the A/PR/8/34 HA with similar efficiency. Analyses of the critical amino acids in the TM site necessary for CTL recognition with the use of synthetic peptides unexpectedly revealed reactivity for the A/PR/8 HA TM site by subtype-specific CTL. This reactivity was only observed with truncated peptides corresponding to a limited portion of the A/PR/8 HA TM site but also required peptide concentrations greater than 10(-7) M. These results suggested either that the endogenously processed A/PR/8 HA TM site generated during infection was larger than the site defined by the truncated cross-reactive peptides or that the concentration of endogenously processed TM site produced during infection was limiting. To distinguish between these possibilities, we expressed in target cells synthetic minigenes encoding only the portion of the A/PR/8 HA transmembrane sites defined by the synthetic peptides. Unlike the peptides, the "preprocessed" endogenous minigene products were not recognized by subtype-specific CTL. These data suggest that the level of available endogenously processed Ag rather than selectivity in the site of fragmentation of newly synthesized Ag may play a critical role in determining whether the complex of the antigenic moiety and class I MHC is efficiently presented to and recognized by class I CTL.     

Isolation and characterization of a CNBr cleavage peptide of influenza viral hemagglutinin stimulatory for mouse cytolytic T lymphocytes

A polypeptide fragment obtained by CNBr cleavage of the hemagglutinin from A/JAPAN/305/57 influenza virus has been purified by using high performance liquid chromatography. The first five N-terminal amino acids as determined by sequential Edman degradations have localized this peptide to the HA2 subunit of the hemagglutinin between residues 103 and 123. This peptide, denoted HA2(103-23), can generate both proliferative and cytolytic responses from spleen cells of BALB/c mice previously immunized with A/JAPAN/305/57. These results demonstrate that a single nonglycosylated fragment of the influenza hemagglutinin as small as 21 amino acid residues is capable of being recognized as an antigenic determinant to generate influenza CTL from primed precursors.     

Rabies virus binding to an acetylcholine receptor alpha-subunit peptide

The binding of 125I-labeled rabies virus to a synthetic peptide comprising residues 173-204 of the alpha 1-subunit of the nicotinic acetylcholine receptor was investigated. Binding of rabies virus to the receptor peptide was dependent on pH, could be competed with by unlabeled homologous virus particles, and was saturable. Synthetic peptides of snake venom, curaremimetic neurotoxins and of the structurally similar segment of the rabies virus glycoprotein, were effective in competing with labeled virus binding to the receptor peptide at micromolar concentrations. Similarly, synthetic peptides of the binding domain on the acetylcholine receptor competed for binding. These findings suggest that both rabies virus and neurotoxins bind to residues 173-204 of the alpha 1-subunit of the acetylcholine receptor. Competition studies with shorter alpha-subunit peptides within this region indicate that the highest affinity virus binding determinants are located within residues 179-192. A rat nerve alpha 3-subunit peptide, that does not bind alpha-bungarotoxin, inhibited binding of virus to the alpha 1 peptide, suggesting that rabies binds to neuronal nicotinic acetylcholine receptors. These studies indicate that synthetic peptides of the glycoprotein binding domain and of the receptor binding domain may represent useful antiviral agents by targeting the recognition event between the viral attachment protein and the host cell receptor, and inhibiting attachment of virus to the receptor.     

Efficient processing of the immunodominant, HLA-A*0201-restricted human immunodeficiency virus type 1 cytotoxic T-lymphocyte epitope despite multiple variations in the epitope flanking sequences

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences.     

Functional constraints of influenza A virus epitopes limit escape from cytotoxic T lymphocytes

Viruses can exploit a variety of strategies to evade immune surveillance by cytotoxic T lymphocytes (CTL), including the acquisition of mutations in CTL epitopes. Also for influenza A viruses a number of amino acid substitutions in the nucleoprotein (NP) have been associated with escape from CTL. However, other previously identified influenza A virus CTL epitopes are highly conserved, including the immunodominant HLA-A*0201-restricted epitope from the matrix protein, M1(58-66). We hypothesized that functional constraints were responsible for the conserved nature of influenza A virus CTL epitopes, limiting escape from CTL. To assess the impact of amino acid substitutions in conserved epitopes on viral fitness and recognition by specific CTL, we performed a mutational analysis of CTL epitopes. Both alanine replacements and more conservative substitutions were introduced at various positions of different influenza A virus CTL epitopes. Alanine replacements for each of the nine amino acids of the M1(58-66) epitope were tolerated to various extents, except for the anchor residue at the second position. Substitution of anchor residues in other influenza A virus CTL epitopes also affected viral fitness. Viable mutant viruses were used in CTL recognition experiments. The results are discussed in the light of the possibility of influenza viruses to escape from specific CTL. It was speculated that functional constraints limit variation in certain epitopes, especially at anchor residues, explaining the conserved nature of these epitopes.