An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections.

A single broadly reactive standard ELISA is commonly applied to control small ruminant lentivirus (SRLV) spread, but type specific ELISA strategies are gaining interest in areas with highly prevalent and heterogeneous SRLV infections. Short (15-residue) synthetic peptides (n=60) were designed in this study using deduced amino acid sequence profiles of SRLV circulating in sheep from North Central Spain and SRLV described previously. The corresponding ELISAs and two standard ELISAs were employed to analyze sera from sheep flocks either controlled or infected with different SRLV genotypes. Two outbreaks, showing SRLV-induced arthritis (genotype B2) and encephalitis (genotype A), were represented among the infected flocks. The ELISA results revealed that none of the assays detected all the infected animals in the global population analyzed, the assay performance varying according to the genetic type of the strain circulating in the area and the test antigen. Five of the six highly reactive (57-62%) single peptide ELISAs were further assessed, revealing that the ELISA based on peptide 98M (type A ENV-SU5, consensus from the neurological outbreak) detected positives in the majority of the type-A specific sera tested (Se: 86%; Sp: 98%) and not in the arthritic type B outbreak. ENV-TM ELISAs based on peptides 126M1 (Se: 82%; Sp: 95%) and 126M2 0,65 0.77 (Se: 68%; Sp: 88%) detected preferentially caprine arthritis encephalitis (CAEV, type B) and visna/maedi (VMV, type A) virus infections respectively, which may help to perform a preliminary CAEV vs. VMV-like typing of the flock. The use of particular peptide ELISAs and standard tests individually or combined may be useful in the different areas under study, to determine disease progression, diagnose/type infection and prevent its spread.

Induction of multiepitopic and long-lasting immune responses against tumour antigens by immunization with peptides, DNA and recombinant adenoviruses expressing minigenes.

The development of immunization strategies to induce strong and multiepitopic T-cell responses against tumour antigens is needed for anti-tumour immunotherapy. However, a common finding after immunization with complex antigens is the preferential induction of immune responses against immunodominant epitopes. In this study, with the aim of inducing multiepitopic responses against several common tumour antigens, we have designed a minigene construct encoding four human leucocyte antigen (HLA)-A2-restricted epitopes belonging to tumour antigens CEA (CEA-691 and CEA-571), MAGE2 (MAGE2-157) and MAGE3 (MAGE3-112), as well as the universal PADRE epitope recognized by T helper lymphocytes. To optimize the activation of immune responses against these epitopes, we have used different antigen formats (short peptides encompassing individual epitopes and DNA plasmids or adenoviral constructs expressing the minigene) in single or combined immunization schedules. A single immunization with either DNA plasmid or recombinant adenovirus induced a monospecific immune response against the immunodominant epitope CEA-571, whereas immunization with the peptide pool induced responses against all epitopes. Combination of peptide priming followed by a boost with the plasmid and the recombinant adenovirus expressing the minigene induced stronger, multi-specific and long-lasting immune responses, overcoming the immunodominance imposed by the main T-cell epitope. Moreover, these combined immunization strategies were able to induce responses that were able to recognize Mel624 HLA-A2+ tumour cells expressing MAGE2. These results suggest that heterologous immunization strategies combining peptides and DNA or recombinant adenoviruses can be useful to broaden the specificity and enhance the efficacy of subunit vaccines.

Monocyte-derived dendritic cells from HCV-infected patients transduced with an adenovirus expressing NS3 are functional when stimulated with the TLR3 ligand poly(I:C).

Dendritic cells (DC) transfected with an adenovirus encoding hepatitis C virus (HCV) NS3 protein (AdNS3) induce potent antiviral immune responses when used to immunize mice. However, in HCV infected patients, controversial results have been reported regarding the functional properties of monocyte-derived DC (MoDC), a cell population commonly used in DC vaccination protocols. Thus, with the aim of future vaccination studies we decided to characterize MoDC from HCV patients transfected with AdNS3 and stimulated with the TLR3 ligand poly(I:C). Phenotypic and functional properties of these cells were compared with those from MoDC obtained from uninfected individuals. PCR analysis showed that HCV RNA was negative in MoDC from patients after the culture period. Also, phenotypic analysis of these cells showed lower expression of CD80, CD86, and CD40, but similar expression of HLA-DR molecules as compared to MoDC from uninfected individuals. Functional assays of MoDC obtained from patients and controls showed a similar ability to activate allogeneic lymphocytes or to produce IL-12 and IL-10, although lower IFN-alpha levels were produced by cells from HCV patients after poly(I:C) stimulation. Moreover, both groups of MoDC induced similar profiles of IFN-gamma and IL-5 after stimulation of allogeneic T-cells. Finally, migration assays did not reveal any difference in their ability to respond to CCL21 chemokine. In conclusion, MoDC from HCV patients are functional after transduction with AdNS3 and stimulation with poly(I:C). These findings suggest that these cells may be useful for therapeutic vaccination in chronic HCV infection.

Characterization of T-cell responses against immunodominant epitopes from hepatitis C virus E2 and NS4a proteins.

Successful clearance of hepatitis C virus (HCV) infection has been associated with strong cellular immune responses against viral antigens. However, although the magnitude of these responses is clearly important for viral eradication, more studies are needed to unravel the fine specificity of the protective anti-HCV immunity in infected patients. This was the aim of the present study. Overlapping peptides spanning the sequence of HCV E2 and NS4a proteins were used to stimulate T cells from patients with chronic hepatitis C divided into three groups: naïve patients, patients who exhibited sustained response to interferon (IFN)-alpha therapy and patients who failed to respond to the treatment. Interleukin-2 production by stimulated cells was measured in each case. Patients with sustained response to therapy had stronger responses to E2 peptides than nonresponders, whereas naïve patients demonstrated intermediate reactivity. In the case of NS4a, responses against peptides where similar in all groups of patients. Analysis of the peptides recognized by T cells showed that responses were broad and heterogeneous, and some immunodominant epitopes, preferentially recognized by patients exhibiting sustained response to treatment, were found. These results confirm the role of cellular immune responses in viral clearance, and stress the importance of immunodominant regions within HCV antigens. These viral sequences may represent valuable immunogens for preparation of therapeutic or prophylactic vaccines.

Carcinoembryonic antigen as a target to induce anti-tumor immune responses.

Identification of relevant targets for cancer therapy is a major goal in cancer research. In this field, the identification of tumor antigens has opened the possibility of inducing specific anti-tumor immune responses. Among these antigens, carcinoembryonic antigen (CEA) is especially relevant because CEA is expressed in a wide variety of adenocarcinomas such as colon, rectum, pancreas, gastric, breast, etc. The present review focuses on different strategies to induce anti-CEA immune responses. In a first group of strategies, the antigen is administered using viral and bacterial vectors expressing CEA, dendritic cells loaded with CEA protein, or dendritic cells transfected with DNA or RNA expressing CEA. A second group of strategies is based on immunizations with antigenic peptide determinants from CEA, rather than with immunogens containing the whole protein. This has been possible due to the identification of different peptide determinants from CEA, which when presented by MHC class I molecules, are recognized by T cytotoxic lymphocytes. More recently, due to the importance of CD4(+) T cells in the induction of immune responses, T helper peptides presented by MHC class II molecules have also been identified. To overcome the poor immunogenicity of CEA-derived peptide determinants, a common feature of self-antigens, their sequence has been modified to improve binding to MHC molecules or recognition by T cell receptors. Finally, in order to enhance immunization efficacy, some of these strategies have combined the administration of immunogens and cytokines or co-stimulatory molecules. Some of the immunization protocols developed are being tested in clinical trials with promising results. Thus, CEA may prove to be a valuable target antigen for the therapy of a high number of malignancies.

T-helper cell response to woodchuck hepatitis virus antigens after therapeutic vaccination of chronically-infected animals treated with lamivudine.

BACKGROUND/AIMS: Immunotherapy of patients chronically-infected with hepatitis B virus (HBV) may have the risk of fulminant hepatitis. This risk might be diminished if immunotherapy was carried out under conditions of low viremia. METHODS: Five woodchucks chronically-infected with woodchuck hepatitis virus (WHV), a virus closely related to HBV, were treated with lamivudine for 23 weeks. At week 10, when viremia had decreased by 3-5 logs, three woodchucks were vaccinated with woodchuck hepatitis virus surface antigen (WHsAg) plus the T-helper determinant FISEAIIHVLHSR. RESULTS: It was found that the administration of lamivudine only, had no effect on the T-helper response against WHV antigens. By contrast, vaccination induced T-helper responses against WHV antigens, shifting the cytokine profile from Th2 to Th0/Th1, but was without effect on viremia, WHsAg levels, or anti-WHs antibodies. Analysis of liver biopsies showed that lamivudine administration may have reduced hepatic inflammation. By contrast, vaccination clearly enhanced hepatic inflammation. After lamivudine withdrawal, viremia returned to high levels. CONCLUSIONS: These results suggest that therapeutic vaccination of chronically-infected woodchucks under conditions of low viremia shifts the cytokine profile against viral antigens towards Th0/Th1. This shift may prevent the efficient induction of anti-WHs antibodies.

Immunization with a tumor-associated CTL epitope plus a tumor-related or unrelated Th1 helper peptide elicits protective CTL immunity.

Immunization with cytotoxic T cell epitope SPSYVYHQF (AH1), derived from MuLV gp70 envelope protein expressed by CT26 tumor cells, does not protect BALB/c mice against challenge with CT26 tumor cells. By contrast, immunization with AH1 plus T helper peptides OVA(323-337) or SWM(106-118) eliciting Th1 and Th0 profiles, protected 83% and 33% of mice, respectively. Interestingly, immunization with AH1 plus both helper peptides reverted the efficacy to 33%. We identified the endogenous T helper peptide p(320-333) from gp70 which elicits a Th1 profile and is naturally processed. As for OVA(323-337), immunization with p(320-333) alone did not protect against tumor challenge. However, p(320-333) plus AH1 protected 89% of mice at day 10 after vaccination. Only 20% of mice vaccinated with AH1 + OVA(323-337) or AH1 + p(320-333) were protected when challenged 80 days after immunization. Treatment with OVA(323-337) or with p(320-333) around established tumors delayed tumor growth. Our results show that tumor-related as well as tumor-unrelated but strong Th1 peptides may be useful for inducing CTL responses in tumor immunotherapy.

Characterization of an immunologically conserved epitope from hepatitis C virus E2 glycoprotein recognized by HLA-A2 restricted cytotoxic T lymphocytes.

BACKGROUND/AIMS: Identification of epitopes recognized by cytotoxic T lymphocytes (CTL) in hepatitis C virus (HCV) proteins is of importance because they can be used for vaccination, treatment of infection or monitoring of immune responses. Our purpose was to characterize new CTL epitopes in HCV structural proteins. METHODS: Peptides were synthesized and tested in HLA-A2 binding assays. Binder peptides were used to stimulate peripheral blood mononuclear cells from HCV+ patients and controls, and activity measured in chromium release and ELISPOT assays. RESULTS: Twenty binder peptides were found, and stimulation of HCV+ patient cells with nine peptides showing high binding ability led to the growth of CD8+ CTL recognizing peptide E2(614-622) in association with HLA-A2. Peptide E2(614-622) was recognized by 30% of HLA-A2+ patients with chronic HCV infection, but no responses were observed in control groups. Five peptides derived from region E2(614-622) from 26 different viral isolates bound to HLA-A2 molecules, and all of them but one, containing Phe at position 622, were recognized by E2(614-622) specific CTL. CONCLUSIONS: These results show that peptide E2(614-622) belongs to a highly conserved region of HCV E2, and might be a good candidate to induce anti-HCV CTL responses in HLA-A2+ subjects.

Respuesta inmunitaria en la infección por el virus de la hepatitis C / Immune response in hepatitis C virus infection

La infección por el virus de la hepatitis C (VHC) se caracteriza por la alta tasa de cronificación viral. Aunque los tratamientos antivirales son eficaces en algunos pacientes, todavía queda un porcentaje alto de ellos que permanece crónicamente infectado. El estudio de los mecanismos del sistema inmunitario involucrados en la eliminación viral es importante tanto para el desarrollo de vacunas como para la comprensión de los fenómenos inmunopatológicos asociados a la infección. Aunque todos los pacientes con infección crónica desarrollan anticuerpos f rente a las proteínas virales, el alto grado de mutación de las regiones reconocidas por anticuerpos neutralizantes hace que la respuesta humoral no sea eficaz. Por otro lado, la eliminación de la infección por VHC tras una hepatitis aguda o tras el tratamiento con IFN- , se asocia con una potente respuesta de los linfocitos T CD4 y CD8, junto con un perfil de citocinas Th1. Sin embargo, la infección crónica viene acompañada con bajas respuestas inmunitarias c e l u l a res, así como un perfil de citocinas de tipo Th2.Además de su alta tasa de mutación, el VHC ha desarrollado mecanismos de interacción con componentes celulare s que desempeñan el normal funcionamiento del sistema inmunitario, permitiendo así la evasión de la respuesta y la cronificación viral. Las futuras vacunas, así como los tratamientos frente al VHC, habrán de tener en cuenta estos factores para conseguir una eliminación eficaz de la infección (AU)

Specific and general HLA-DR binding motifs: comparison of algorithms.

Using panels of peptides well characterized for their ability to bind to HLA DR1, DRB1*1101, or DRB1*0401 molecules, algorithms were deduced to predict binding to these molecules. These algorithms consist of blocks of 8 amino acids containing an amino acid anchor (Tyr, Phe, Trp, Leu, Ile, or Val) at position i and different amino acid combinations at positions i+2 to i+7 depending on the class II molecule. The sensitivity (% of correctly predicted binder peptides) and specificity (% of correctly predicted non-binder peptides) of these algorithms, were tested against different independent panels of peptides and compared to other algorithms reported in the literature. Similarly, using a panel of 232 peptides able to bind to one or more HLA molecules as well as 43 non-binder peptides, we deduced a general motif for the prediction of binding to HLA-DR molecules. The sensitivity and specificity of this general motif was dependent on the threshold score used for the predictions. For a score of 0.1, the sensitivity and specificity were 84.7% and 69.8%, respectively. This motif was validated against several panels of binder and non-binder peptides reported in the literature, as well as against 35, 15-mer peptides from hepatitis C virus core protein, that were synthesized and tested in a binding assay against a panel of 19 HLA-DR molecules. The sensitivities and specificities against these panels of peptides were similar to those attained against the panels used to deduce the algorithm. These results show that comparison of binder and non-binder peptides, as well as correcting for the relative abundance of amino acids in proteins, is a useful approach to deduce performing algorithms to predict binding to HLA molecules.

Adenoviral gene transfer of interleukin 12 into tumors synergizes with adoptive T cell therapy both at the induction and effector level.

Tumors infected with a recombinant defective adenovirus expressing interleukin 12 (IL-12) undergo regression, associated with a cytotoxic T lymphocyte (CTL)-mediated antitumor immune response. In the present study we generated anti-CT26 CTLs by short-term coculture of CT26 cells and lymph node cells obtained from mice harboring subcutaneous CT26 tumors injected with an adenoviral vector expressing IL-12 (AdCMVIL-12), control adenovirus (AdCMVlacZ), or saline. Regression of small intrahepatic CT26 tumors in unrelated syngeneic animals was achieved with CTLs derived from mice whose subcutaneous tumors had been injected with AdCMVIL-12 but not with CTLs from the other two control groups. The necessary and sufficient effector cell population for adoptive transfer consisted of CD8+ T cells that showed anti-CT26 specificity partly directed against the AH1 epitope presented by H-2Ld. Interestingly, treatment of a subcutaneous tumor nodule with AdCMVIL-12, combined with intravenous adoptive T cell therapy with short-term CTL cultures, had a marked synergistic effect against large, concomitant live tumors. Expression of IL-12 in the liver in the vicinity of the hepatic tumor nodules, owing to spillover of the vector into the systemic circulation, appeared to be involved in the increased in vivo antitumor activity of injected CTLs. In addition, adoptive T cell therapy improved the outcome of tumor nodules transduced with suboptimal doses of AdCMVIL-12. Our data provide evidence of a strong synergy between gene transfer of IL-12 and adoptive T cell therapy. This synergy operates both at the induction and effector phases of the CTL response, thus providing a rationale for combined therapeutic strategies for human malignancies.

T(h)1 but not T(h)0 cell help is efficient to induce cytotoxic T lymphocytes by immunization with short synthetic peptides.

Immunization of BALB/c mice with peptide HVSGHRMAWDMMMNWA, encompassing residues 121-135 from hepatitis C virus E1 protein, induced CD4(+) T(h)1 cells as well as a long-lasting CD8(+) cytotoxic T lymphocyte (CTL) response in vivo when the peptide was administered s.c. with or without incomplete Freund's adjuvant. Using truncated peptides from this sequence it was shown that the determinant recognized by cytotoxic T cells was encompassed by residues SGHRMAWDM. Deletion of residues from the N-terminus or the C-terminus of the wild-type peptide abrogated its helper character. When Val122 of the wild peptide was replaced by Ala, the ability to induce a cytotoxic response was lost concomitantly with the loss of the T(h)1 pattern of cytokine production. Interestingly, the Ala-modified peptide, when co-immunized with a peptide encompassing residues 323-329 from ovalbumin (OVA), which is able to induce a T(h)1 response in BALB/c mice, restored the capacity of the modified peptide to induce CTL. However, co-immunization of the Ala-modified peptide with a peptide encompassing residues 106-118 from sperm whale myoglobin, which induces a T(h)0 cytokine profile in BALB/c mice, was much less efficient than the OVA peptide to restore CTL induction. These results demonstrate that CTL induction with a short synthetic peptide requires that this peptide contains domains recognized by T(c) cells as well as by T(h)1 cells. For those peptides that do not contain this type of T(h) domain, competent T cell help can be provided by co-immunization with a distinct peptide that is able to stimulate a T(h)1 response.

Different doses of adenoviral vector expressing IL-12 enhance or depress the immune response to a coadministered antigen: the role of nitric oxide.

Joint immunization with two recombinant adenoviruses, one expressing hepatitis C virus (HCV) core and E1 proteins and another expressing IL-12 (RAdIL-12), strongly potentiates cellular immune response against HCV Ags in BALB/c mice when RAdIL-12 was used at doses of 1 x 105-1 x 107 plaque-forming units. However, cellular immunity against HCV Ags was abolished when higher doses (1 x 108 plaque-forming units) of RAdIL-12 were used. This immunosuppressive effect was associated with marked elevation of IFN-gamma and nitric oxide in the serum and increased cell apoptosis in the spleen. Administration of N-nitro-L -arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, to mice that received high doses of RAdIL-12 was lethal, whereas no apparent systemic toxicity by L -NAME was observed in those immunized with lower doses of the adenovirus. Interestingly, in mice immunized with recombinant adenovirus expressing core and E1 proteins of HCV in combination with RAdIL-12 at low doses (1 x 107 plaque-forming units), L -NAME inhibited T cell proliferation and CTL activity in response to HCV Ags and also production of Abs against adenoviral proteins. In conclusion, gene transfer of IL-12 can increase or abolish cell immunity against an Ag depending of the dose of the vector expressing the cytokine. IL-12 stimulates the synthesis of NO which is needed for the immunostimulating effects of IL-12, but apoptosis of T cells and immunosuppression ensues when IFN-gamma and NO are generated at very high concentrations.

Cellular immunity to hepatitis C virus core protein and the response to interferon in patients with chronic hepatitis C.

To investigate the involvement of T-cell response against hepatitis C virus (HCV) antigens in viral clearance after interferon therapy, we measured interleukin-2 (IL-2) production by peripheral mononuclear cells in response to HCV core in patients with chronic hepatitis C. In a cohort of 43 patients, we investigated the frequency of circulating core-specific T-helper (Th) cell precursors by the limiting-dilution assay, and in a second cohort of 60 patients, we analyzed the response to specific core epitopes using 52 synthetic 15-mer overlapping peptides. We observed that the frequency of core-specific Th cell precursors was significantly higher in patients with sustained biochemical and virological response (SR) after interferon (IFN) therapy (median, 1/55,736) than in untreated patients (1/274,023) or that in patients who remained viremic after completion of the treatment-nonresponders (NR) plus transient responders (TR) (1/1,909,972). Patients who failed to respond to IFN (NR) and those who relapsed after IFN discontinuation (TR) had a similarly low number of precursors. The number of core peptides recognized by SR, TR, NR, UT, and healthy controls was 8.2 +/- 1.5, 6.5 +/- 1.2, 2.0 +/- 0.5, 2.7 +/- 0.9, and 0.3 +/- 0.2, respectively. In SR, the intensity of the proliferative response to core peptides as estimated by the summation of stimulation indexes (sigmaSI) was significantly higher than in NR and than in UT, but not different from that of TR. Our results indicate that both expansion of HCV-specific Th cell precursors and Th cell recognition of multiple core epitopes seem to be important in the elimination of HCV after IFN therapy.

Therapeutic vaccination of woodchucks against chronic woodchuck hepatitis virus infection.

BACKGROUND/AIMS: Therapeutic vaccination is a new approach to treat patients with chronic hepatitis B virus infection. We have used the woodchuck model to examine the efficacy and safety of this approach. METHODS: Seven woodchucks chronically infected with woodchuck hepatitis virus were immunized with surface antigen from this virus, purified from plasma, in conjunction with a peptide named FIS (encompassing amino acids 106-118: FISEAIIHVLHSR from sperm whale myoglobin), which is recognized by T helper lymphocytes. As controls, two woodchucks chronically infected with woodchuck hepatitis virus were immunized: one with FIS only and the other with surface antigen only. RESULTS: Co-immunization with surface antigen and FIS, but not with FIS or surface antigen alone, induced anti-surface antibodies in 7/7 immunized woodchucks. In the two woodchucks in which the highest titer of anti-surface antibody was elicited, severe liver damage was observed: one died of fulminant hepatitis and the other became seriously ill with hepatic injury and had to be sacrificed. CONCLUSIONS: Co-immunization of chronically infected woodchucks with surface antigen and a peptide recognized by T helper cells produces a good anti-surface antibody response. However, this strategy needs to be optimized before its implementation in humans. Although our experiments are not strictly comparable to vaccination of chronically hepatitis B virus-infected patients with recombinant or plasma-derived vaccines, we believe that precautions should be taken to avoid the risk of severe liver injury when immunizing hepatitis B virus carriers.

Induction of cytotoxic T-cell response against hepatitis C virus structural antigens using a defective recombinant adenovirus.

A replication-defective recombinant adenovirus (RAd), RAdCMV-CE1, containing core and E1 genes of hepatitis C virus (HCV) was constructed. RAdCMV-CE1 was able to express core and E1 proteins both in mice and human cells. Immunization of BALB/c mice with RAdCMV-CE1 induced a specific cytotoxic T-cell response against the two HCV proteins. This response was characterized using a panel of 60 synthetic 14- or 15-mer overlapping peptides (10 amino-acid overlap) spanning the entire sequence of these proteins. Five main epitopes were found in the core protein, four of which had been previously described either in mice or humans. One single novel epitope was found in E1. Fine mapping of this E1 determinant, showed that octamer GHRMAWDM is the minimal epitope recognized by cytotoxic T lymphocytes (CTL). The cytotoxic T-cell response was H-2d restricted, lasted for at least 100 days, and was mediated by T cells with the classic CD4-CD8+ phenotype. This work demonstrates that replication-defective recombinant adenoviruses can efficiently express HCV proteins and are able to induce an in vivo cytotoxic T-cell response against a diversity of epitopes from HCV antigens. These vectors should be taken into consideration in the design of vaccines and also as a means to stimulate specific T-cell responses in chronic HCV carriers.

Binding analysis of 95 HIV gp120 peptides to HLA-DR1101 and -DR0401 evidenced many HLA-class II binding regions on gp120 and suggested several promiscuous regions.

To identify HLA-DR-binding peptides within the human immunodeficiency virus (HIV)-1 proteins. 95 overlapping synthetic peptides representing the entire sequence of gp120-LAI were screened for their capacity to bind to two HLA-DR molecules with distant sequences (DR0401 and DR1101). By using a cell surface competitive binding assay, 56 DR-binding peptides were identified, of which 35 bound to both DR1101 and DR0401. A highly significant concordance was evidenced by statistical analysis between binding of peptides to one and to the other DR molecule, suggesting a high proportion of promiscuity among gp120 peptides, even though no clear sequence pattern accounting for such promiscuity was found. DR-binding peptides were located along the entire gp120 sequence. Yet, the majority of them (42 among 56) were concentrated in seven multiagretopic regions that were arbitrarily defined as regions containing four or more overlapping continuous peptides binding to DR1011 and/or DR0401. A good correlation was found between DR-binding regions or DR-binding peptides defined in this study and promiscuous T helper gp120 epitopes previously described in seropositive individuals. All these results suggest that the identification of multiagretopic DR-binding regions may be a great help for the predicition of protein determinants that have the likelihood of being promiscuous T helper epitopes in humans.

Fine analysis of immunoreactivity of V3 peptides: antibodies specific for V3 domain of laboratory HIV type 1 strains recognize multiple V3 sequences synthesized from field HIV type 1 isolates.

Production of cross-reactive antibodies recognizing the V3 loop--that is, the principal neutralizing determinant (PND)--of various HIV-1 isolates is an important challenge in the development of passive immunotherapy or vaccinations against AIDS. We have produced two types of antibodies to the V3 domain of HIV-1: (1) antibodies against the HIV-1 MN laboratory strain generated in rabbits and (2) antibodies targeted to the HIV-1 LAI laboratory strain induced in chimpanzees. These antibodies were shown to be specific for HIV-1 subtype B. The cross-reactivity of these antibodies has been evaluated against a large panel of peptides representing different parts of the V3 loop. Seventy-five peptides, referred to as clinical peptides, were synthesized according to HIV-1 sequences recovered from PMBCs of 27 patients followed in three Parisian hospitals. Thirteen V3 peptides derived from 4 HIV-1 laboratory strains (MN, LAI, SF2, and RF) were also included in the study. The results show that both the amino-terminal and central parts of the V3 loop are immunogenic. The rabbit antibodies against the amino-terminal end of the PND proved to be highly cross-reactive against the clinical peptides. The anti-gp160 antibodies induced in one chimpanzee recognized a significant proportion of the panel of V3 clinical sequences. These antibodies cross-reacted mainly with the apex of the V3 loop. These data give some additional indications on the immunogenicity of the V3 loop and further demonstrate that extensive cross-reactivity of anti-V3 antibodies can be obtained on field HIV-1 isolates despite the high variability of the V3 loop amino acid sequence.

Binding of ALA-substituted analogs of HA306-320 to DR1101, DR1301, and DR0402 molecules: correlation of DR-peptide interactions with recognition by a single TCR.

We tested the hypothesis that a cross-reactive T-cell clone could recognize HA306-320 peptide complexed to autologous HLA-DR1101, and also to allogenic HLA-DR0402 and HLA-DR1301 molecules, because of similar orientations of HA306-320 side chains in the groove of the three DR molecules. To approach peptide orientations in each HLA groove we compared the capacity of Ala-monosubstituted analogs to bind and be presented by DR1101, DR0402, and DR1301. Results indicated that the orientation of HA306-320 in DR1101 was grossly similar to the known orientation of HA307-319 in DR0101. Data suggested many similarities in peptide orientations in DR0402 and DR1301 as well. However, differences in binding were also observed. Ala substitution of Y309 had much less effect on peptide binding to DR1301 and DR0402 than to DR1101 and Ala-substitution of T314 increased affinity for DR1301 but not for DR1101 and DR0402. These alterations of peptide-DR interactions were probably communicated to the upper peptide surface. Indeed, the levels of T-cell clone reactivities against analogs mutated at positions predicted to face the TCR were lower when complexed to allogeneic DR molecules than when complexed to DR1101. Yet these epitopic alterations are likely subtle, since the decreased reactivity of the clone to allogeneic molecules could be compensated by peptide substitution at Y309, predicted to face the MHC.

Production of interleukin-2 in response to synthetic peptides from hepatitis C virus E1 protein in patients with chronic hepatitis C: relationship with the response to interferon treatment.

BACKGROUND/AIMS: The role of cellular immunity in the clearance of hepatitis C virus after interferon therapy has not yet been elucidated. Here, we analyzed the T cell response to peptides from hepatitis C virus E1 protein in untreated and interferon-treated patients with chronic hepatitis C virus infection. METHODS: We used thirty-six 15-mer synthetic peptides from hepatitis C virus E1 protein (genotype 1a) in a sensitive interleukin-2 production assay in two groups of controls (healthy seronegative individuals and patients with liver diseases unrelated to hepatitis C virus), and three groups of patients with chronic hepatitis C: nine patients who cleared the virus after interferon treatment (group 1), nine patients who failed to respond to the therapy (group 2) and nine previously untreated patients (group 3). RESULTS: None of the controls responded to any of the peptides tested, whereas 8/9 (88%) of patients from group 1 responded positively. In contrast, only 2/9 (22%) of patients from group 2 showed peptide recognition. In group 3, 5/9 patients (55%) displayed positive response against E1 peptides. When E1 peptides from the sequence corresponding to genotype 1b (the commonest in patients who were non-responders to interferon) were tested in nine additional interferon-resistant patients (group 2*) a positive response was detected in only three of them (33%). CONCLUSIONS: T cell recognition of hepatitis C virus E1 peptides in patients with chronic hepatitis C who exhibit sustained response to interferon therapy is increased as compared with interferon-resistant cases, suggesting that T cell immunity to hepatitis C virus structural proteins may play a role in the clearance of this viral infection.