Definition of an 18-mer Synthetic Peptide Derived from the GB virus C E1 Protein as a New HIV-1 Entry Inhibitor.

BACKGROUND: A slower progression of AIDS and increased survival in GBV-C positive individuals, compared with GBV-C negative individuals has been demonstrated; while the loss of GBV-C viremia was closely associated with a rise in mortality and increased progression of AIDS. Following on from the previous reported studies that support the thesis that GBV-C E2 interferes with HIV-1 entry, in this work we try to determine the role of the GBV-C E1 protein in HIV-1 inhibition. METHODS: The present work involves the construction of several overlapping peptide libraries scanning the GBV-C E1 protein and the evaluation of their anti-HIV activity. RESULTS: Specifically, an 18-mer synthetic peptide from the GBV-C E1 protein, E1(139-156), showed similar antiviral activity against HIVs from viruses from clades A, B, C, D and AE. Competitive ELISA using specific gp41-targeting mAbs, fluorescence resonance energy transfer as well as haemolysis assays demonstrated that this E1 peptide sequence interacts with the highly conserved N-terminal region of the HIV-1 gp41 (the fusion peptide) which is essential for viral entry. CONCLUSIONS: We have defined a novel peptide lead compound and described the inhibitory role of a highly conserved fragment of the E1 protein. GENERAL SIGNIFICANCE: The results together allow us to consider the non-pathogenic E1 GBV-C protein as an attractive source of peptides for the development of novel anti-HIV therapies.

Surface behavior of peptides from E1 GBV-C protein: Interaction with anionic model membranes and importance in HIV-1 FP inhibition.

The interaction between a peptide sequence from GB virus C E1 protein (E1P8) and its structural analogs (E1P8-12), (E1P8-13), and (E1P8-21) with anionic lipid membranes (POPG vesicles and POPG, DPPG or DPPC/DPPG (2:1) monolayers) and their association with HIV-1 fusion peptide (HIV-1 FP) inhibition at the membrane level were studied using biophysical methods. All peptides showed surface activity but leakage experiments in vesicles as well as insertion kinetics in monolayers and lipid/peptide miscibility indicated a low level of interaction: neither E1P8 nor its analogs induced the release of vesicular content and the exclusion pressure values (πe) were clearly lower than the biological membrane pressure (24-30 mN m(-1)) and the HIV-1 FP (35 mN m(-1)). Miscibility was elucidated in terms of the additivity rule and excess free energy of mixing (GE). E1P8, E1P8-12 and E1P8-21 (but not E1P8-13) induced expansion of the POPG monolayer. The mixing process is not thermodynamically favored as the positive GE values indicate. To determine how E1 peptides interfere in the action of HIV-1 FP at the membrane level, mixed monolayers of HIV-1 FP/E1 peptides (2:1) and POPG were obtained. E1P8 and its derivative E1P8-21 showed the greatest HIV-1 FP inhibition. The LC-LE phase lipid behavior was morphologically examined via fluorescence microscopy (FM) and atomic force microscopy (AFM). Images revealed that the E1 peptides modify HIV-1 FP-lipid interaction. This fact may be attributed to a peptide/peptide interaction as indicated by AFM results. Finally, hemolysis assay demonstrated that E1 peptides inhibit HIV-1 FP activity.

[Does medication abuse in patients with chronic migraine influence the effectiveness of preventive treatment with topiramate?]. / ¿El abuso de farmacos en pacientes con migraña cronica influye en la efectividad del tratamiento preventivo con topiramato?

INTRODUCTION: Patients with chronic migraine (CM) and medication abuse are difficult to treat, and have a greater tendency towards chronification and a poorer quality of life than those with other types of headache. AIM: To evaluate whether the presence of medication abuse lowers the effectiveness of topiramate. PATIENTS AND METHODS: A series of patients with CM were grouped according to whether they met abuse criteria or not. They were advised to stop taking the drug that they were abusing. Treatment was adjusted to match their crises and preventive treatment with topiramate was established from the beginning. The number of days with headache and intense migraine in the previous month and at four months of treatment was evaluated. RESULTS: In all, 262 patients with CM criteria were selected and 167 (63.7%) of them fulfilled abuse criteria. In both groups there was a significant reduction in the number of days with headache/month and number of migraine attacks/month at the fourth month of treatment with topiramate. The percentage of reduction in the number of days with headache/month in CM without abuse was 59.3 ± 36.1%, and with abuse, 48.7 ± 41.7% (p = 0.0574). The percentage of reduction in the number of days with intense migraine/month in CM without abuse was 61.2%, and with abuse, 50% (p = 0.0224). Response rate according to the number of days with headache/month in CM without abuse was 69%, and with abuse, 57%. Response rate according to the number of intense migraines/month in CM without abuse was 76.8%, and in CM with abuse, 61% (p = 0.0097). CONCLUSIONS: Topiramate was effective in patients with CM with and without medication abuse, although effectiveness is lower in the latter case.

TITLE: El abuso de farmacos en pacientes con migraña cronica influye en la efectividad del tratamiento preventivo con topiramato?Introduccion. Los pacientes con migraña cronica (MC) y abuso de medicacion son dificiles de tratar y tienen peor calidad de vida que otros pacientes con migrañas. Objetivo. Valorar si la presencia de abuso de farmacos disminuye la efectividad del topiramato. Pacientes y metodos. Una serie de pacientes con MC fueron agrupados segun presentasen criterios de abuso o no abuso de farmacos. Se les aconsejo la supresion del farmaco del cual abusaban. Se ajusto el tratamiento de sus crisis y se inicio tratamiento preventivo desde el principio con topiramato. Se valoro el numero dias con cefalea y migrañas intensas en el mes previo y al cuarto mes de tratamiento. Resultados. Fueron seleccionados 262 pacientes con criterios de MC, y de ellos 167 (63,7%) cumplieron criterios de abuso. En ambos grupos hubo una reduccion significativa del numero de dias con cefalea/mes y numero de crisis de migraña/mes al cuarto mes de tratamiento con topiramato. Porcentaje de reduccion de dias con cefalea/mes en MC sin abuso, 59,3 ± 36,1%; y con abuso, 48,7 ± 41,7% (p = 0,0574). Porcentaje de reduccion de migrañas intensas/mes en MC sin abuso, 61,2%; y con abuso, 50% (p = 0,0224). Tasa de respondedores segun numero de dias con cefalea/mes en MC sin abuso, 69%; y con abuso, 57%. Tasa de respondedores segun numero de migrañas intensas/mes en MC sin abuso, 76,8%; y en MC con abuso, 61% (p = 0,0097). Conclusiones. El topiramato fue efectivo en pacientes con MC sin y con abuso de farmacos, aunque con menor efectividad en estos ultimos.

[A study to compare independent groups of patients with episodic migraine who were treated preventively with flunarizine or nadolol]. / Estudio comparativo de grupos independientes de pacientes con migrana episodica tratados preventivamente con flunaricina o nadolol.

INTRODUCTION. Flunarizine, with level of evidence A, and nadolol, with evidence level C, would be indicated as preventive treatment of migraine. Yet, no previous studies have been conducted to compare the effectiveness of the two drugs. AIM. To compare the effectiveness parameters in independent groups of patients treated preventively with one of the pharmaceuticals from the study, the same protocol being applied in both cases. PATIENTS AND METHODS. The subjects selected for the study were patients with episodic migraine (according to 2004 International Headache Society criteria) who had undergone preventive treatment for the first time, with flunarizine (5 mg/day) or nadolol (20-40 mg/day). The main effectiveness variables (reduction in the number of seizures at four months of treatment and responder rates) were analysed. RESULTS. The study included 227 patients who intended to receive treatment: 155 with flunarizine (80.5% females; mean age: 38.3 ± 12.1 years) and 72 with nadolol (63.8% females; mean age: 37.1 ± 12.0 years). The mean number of seizures prior to treatment was 6.09 ± 2.6 in the flunarizine group and 5.1 ± 1.7 in the nadolol group (p = 0.0079); at four months of treatment it was 2.61 ± 2.4 in the flunarizine group and 2.77 ± 2.4 in the nadolol group (p = NS). Percentage of reduction of migraines: 55.2% with flunarizine and 50.4% with nadolol (p = NS). The responder rate was 69% with flunarizine and 67% with nadolol (p = NS). The excellent response rate (reduction in the number of seizures by 75% or more) was 52.2% with flunarizine and 36.1% with nadolol (p = 0.0077). Percentage of adverse side effects: 48.3% with flunarizine and 25% with nadolol (p = 0.0009). The satisfaction rate was similar in both groups, 68%. CONCLUSIONS. Both flunarizine and nadolol proved to be effective in the preventive treatment of episodic migraine. Flunarizine is used more often in our milieu and was less well tolerated.

Study of the inhibition capacity of an 18-mer peptide domain of GBV-C virus on gp41-FP HIV-1 activity.

The peptide sequence (175-192) RFPFHRCGAGPKLTKDLE (P59) of the E2 envelope protein of GB virus C (GBV-C) has been proved to decrease cellular membrane fusion and interfere with the HIV-1 infectivity in a dose-dependent manner. Based on these previous results, the main objective of this study was to deepen in the physicochemical aspects involved in this interaction. First, we analyzed the surface activity of P59 at the air-water interface as well as its interaction with zwitterionic or negatively charged lipid monolayers. Then we performed the same experiments with mixtures of P59/gp41-FP. Studies on lipid monolayers helped us to understand the lipid-peptide interaction and the influence of phospholipids on peptide penetration into lipid media. On another hand, studies with lipid bilayers showed that P59 decreased gp41-FP binding to anionic Large Unilamellar Vesicles. Results can be attributed to the differences in morphology of the peptides, as observed by Atomic Force Microscopy. When P59 and gp41-FP were incubated together, annular structures of about 200 nm in diameter appeared on the mica surface, thus indicating a peptide-peptide interaction. All these results confirm the gp41-FP-P59 interaction and thus support the hypothesis that gp41-FP is inhibited by P59.

[Patients with headache and medication abuse. Indicators of response to ambulatory treatment]. / Pacientes con cefalea y abuso de medicación. Indicadores de respuesta al tratamiento ambulatorio.

INTRODUCTION: Patients with headache and medication abuse (HMA) are difficult to treat, have a greater tendency towards chronification and a poorer quality of life than those with other types of headache. AIM. To evaluate the indicators showing that these patients are responding to ambulatory treatment. PATIENTS AND METHODS: From a series of patients with migraine, we selected those who satisfied HMA criteria according to the appendix of the 2006 International Classification of the Headache Disease (ICHD-2) and who had never previously undergone treatment. As outpatients, they were advised to stop taking the drug that they were abusing. The treatment of their seizures was adjusted with the most efficient drugs and preventive treatment was started from the outset with topiramate or flunarizine. Patients were grouped according to whether they continued with HMA or not. Comparisons were made between the number of days with headache during the previous month and after four months of treatment and the persistence of abuse. RESULTS: HMA criteria were met by 178 patients (mean age 40.9; 88.7% females). Results showed that 68.5% (122 patients) responded and no longer met HMA criteria after treatment. The treatment used for their seizures (triptans, nonsteroidal antiinflammatory drugs, analgesics) and preventive treatment (topiramate or flunarizine) were similar in both groups. The average number of days with headache prior to treatment was 18.52 in the group that responded and 20.87 (p = 0.0263) in the group that did not respond to treatment. In the group of responders 7.3% dropped out of preventive treatment compared with 35% (p = 0.0001) in the group of non-responders. CONCLUSIONS: A higher number of days with headache during the previous month and withdrawing from preventive treatment were indicators of a bad progression.

Application of a chimeric synthetic peptide in the development of a serologic method for the diagnosis of hepatitis G virus infection.

New putative antigenic peptides corresponding to the N- and C-terminal of the E2 envelope protein of GBV-C/HGV were synthesized using solid-phase chemistry. The antigens were obtained in linear and chimeric forms with the main aim of improving the sensitivity of the enzyme immunoassays. Furthermore, CD and FTIR have been used in conjunction to characterize their conformational changes showing that the chimeric peptide presents a more ordered secondary structure than its parent peptides.

[Application of a clinical pathway for the treatment of transient ischaemic attacks: implementation strategies and appraisal at two years]. / Aplicación de una vía clínica para el tratamiento del accidente isquémico transitorio: estrategias de implantación y evaluación a los dos años.

INTRODUCTION: Management of cerebral vascular pathologies by means of clinical pathways allows us to make cost effective use of resources, to enhance health care quality and to obtain a greater degree of satisfaction in patients. AIMS: To assess the efficiency of applying a clinical pathway designed for the treatment of transient ischemic attacks (TIA) by monitoring a series of indicators that enable us to detect existing problems, to introduce any corrections that are needed and to draw conclusions that can be useful in the future. PATIENTS AND METHODS: To this end, a clinical pathway was drawn up with the general agreement of the members of our service and the different professionals involved in caring for these patients. Analyses were performed to study the data from 1998 to 2001, prior to implementation of the pathway, and from 2002 and 2003, which were the first years in which it was being applied. Altogether 1,433 patients with a diagnosis of TIA were hospitalised during this period, 554 of whom were admitted during the years 2002 and 2003. RESULTS: Of this group, the pathway was initially applied in 123 cases and 62 completed it. The mean stay in hospital was reduced from 9.2 days in 2000 to 5.7 days in 2003. The mean stay of patients who fulfilled all the requirements of the pathway was only 2.9 days. As far as the survey on satisfaction is concerned, 97% of patients said they were satisfied or very satisfied with the care they had received. CONCLUSIONS: The application of a clinical pathway in the treatment of TIA resulted in a high degree of satisfaction among the patients who were treated and a notable reduction in the mean stay in hospital.

[Sexual dysfunction management in multiple sclerosis]. / Abordaje de la disfunción sexual en la esclerosis múltiple.

INTRODUCTION: Sexual dysfunction is a frequent disorder associated to multiple sclerosis, that contributes to the worsening of life quality of these patients. AIM: To ascertain how it is managed in a demyelinating disease unit. PATIENTS AND METHODS: It was done an anonymous poll to multiple sclerosis patients in a demyelinating disease unit. The following variables were analysed: age, sex, marital status, education degree, sexual dysfunction, vesical dysfunction, gait disturbances and duration of illness. RESULTS: 67 of 97 patients answered. 74.6% females. Average age was 43.7 years. Average developing time was 11.3 years. 58% of the patients had vesical dysfunction. 43% had sexual dysfunction. There was relation with statistical significance between sexual and vesical dysfunction but not among the rest of variables. 63% of the patients with sexual dysfunction had never talked about this problem with their doctors. That the patient talked about sexual dysfunction was related with the fact that the neurologist asked for or not, and if sexual dysfunction was an important problem for the patient. The neurologist had asked for dysfunction sexual symptoms to 30% of the patients, and this was more frequent if the patient was male and if he or she had gait disturbances. CONCLUSIONS: Sexual dysfunction is a frequent and important problem for patients with multiple sclerosis. According to our results, this problem is raised up in an insufficient manner as much for patients as for neurologists.

Abordaje de la disfunción sexual en la esclerosis múltiple / Sexual dysfunction management in multiple sclerosis

Introducción. La disfunción sexual es un trastorno frecuente asociado a la esclerosis múltiple (EM) y contribuye al empeoramiento de la calidad de vida de estos pacientes. Objetivo. Valorar cómo se aborda este tema en una unidad de enfermedades desmielinizantes. Pacientes y métodos. Encuesta de respuesta anónima a pacientes con EM de una unidad de enfermedades desmielinizantes. Variables analizadas: edad, sexo, estado civil, nivel de escolarización, disfunción sexual, disfunción vesical, trastornos de la marcha y tiempo de evolución. Análisis bivariante y regresión logística. Resultados. Respondieron 67 de 97 pacientes. El 74,6% eran mujeres. Edad media: 43,7 años. Tiempo medio de evolución: 11,3 años. El 58% tenía disfunción vesical. La disfunción sexual estaba presente en el 43% de los pacientes. Observamos relación estadísticamente significativa entre la disfunción sexual y la disfunción vesical, pero no con el resto de las variables analizadas. El 63% de los pacientes con disfunción sexual no había comentado nunca este problema con el neurólogo. Las variables que se relacionan de forma independiente con que el paciente hable con el especialista de disfunción sexual son que se le interrogue a este respecto y que sea un problema importante en relación con su enfermedad. El neurólogo había preguntado por síntomas de disfunción sexual al 30% de los pacientes y el hecho de preguntar se relacionó con que el paciente fuera varón y con que tuviera trastornos en la marcha. Conclusión. La disfunción sexual es un problema frecuente e importante para los pacientes con EM. Según nuestros resultados, se aborda de manera insuficiente

Introduction. Sexual dysfunction is a frequent disorder associated to multiple sclerosis, that contributes to the worsening of life quality of these patients. Aim. To ascertain how it is managed in a demyelinating disease unit. Patients and methods. It was done an anonymous poll to multiple sclerosis patients in a demyelinating disease unit. The following variables were analysed: age, sex, marital status, education degree, sexual dysfunction, vesical dysfunction, gait disturbances and duration of illness. Results. 67 of 97 patients answered. 74.6% females. Average age was 43.7 years. Average developing time was 11.3 years. 58% of the patients had vesical dysfunction. 43% had sexual dysfunction. There was relation with statistical significance between sexual and vesical dysfunction but not among the rest of variables. 63% of the patients with sexual dysfunction had never talked about this problem with their doctors. That the patient talked about sexual dysfunction was related with the fact that the neurologist asked for or not, and if sexual dysfunction was an important problem for the patient. The neurologist had asked for dysfunction sexual symptoms to 30% of the patients, and this was more frequent if the patient was male and if he or she had gait disturbances. Conclusions. Sexual dysfunction is a frequent and important problem for patients with multiple sclerosis. According to our results, this problem is raised up in an insufficient manner as much for patients as for neurologists

Synthetic peptides for the immunodiagnosis of human diseases.

Synthetic peptides have been shown to be valuable tools for viral laboratory diagnosis and can provide uniform, chemically well-defined antigens for antibody analysis, reducing inter- and intra-assay variation. The main aim in the development of peptide-based diagnostic tests is to recognise specific antibodies induced by the whole viral proteins but using selected short fragments containing the most potent antigenic determinants. The success of this approach depends on the extent to which synthetic peptides are able to mimic the immunodominant epitopes of antigens. In recent years, synthetic peptides that mimic specific epitopes of infectious agents' proteins have been used in diagnostic systems for various human diseases. The present review summarizes some of the drawbacks of the use of relatively short linear peptides as antigenic substrates and the subsequent chemical strategies developed in order to overcome the low peptide reactivity against specific antibodies. Moreover, it outlines the most significant bibliography published in the last five years which provides validated peptide based tests potentially useful for diagnosis of viral, bacterial, parasitic and autoimmune diseases.

The covalent coupling of HAV-VP3 (110-121) synthetic peptide to liposomes: physicochemical studies.

In this work we have studied the conjugation of the immunogenic peptide sequence (110-121) belonging to the VP3 capsid protein of hepatitis A virus to the surface of preformed liposomes by means of an amide bond between the vesicles and the synthetic peptide. The surface activity of the conjugate at air/water interface was determined. Moreover, the interaction of the conjugate with lipids was also studied recording the pressure increases produced after the injection of the liposome-peptide preparation under dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and stearylamine (SA) monolayers at different initial surface pressures. As expected, due to the negative net charge of the liposome-peptide complex, the higher interaction was found with positive charge monolayers (SA). However, the conjugate was also able to incorporate to zwitterionic and anionic lipids. This behaviour was also confirmed performing compression isotherms of monolayers of these lipids spread on subphases containing the conjugate. These results suggest that the coupling of VP3 (110-121) to liposomes does not influence its ability to interact with membrane lipids such as DPPC and DPPG. Then it can be assumed that its immunogenicity will be preserved or even increased after this modification. All these results are also useful in the preparation of liposome-based synthetic peptide vaccines.

Lipophilic derivatization of synthetic peptides belonging to NS3 and E2 proteins of GB virus-C (hepatitis G virus) and its effect on the interaction with model lipid membranes.

The synthesis by solid-phase methodologies of peptides belonging to structural and non-structural proteins of GB virus C as well as its N-alpha-acylation with myristate and palmitate fatty acids is described. To explore the peptide-lipid interactions we have used liposomes composed of dipalmitoylphosphatidylcholine as model membranes and complementary spectroscopic and calorimetric techniques. Our results show that structural and more clearly the structural lipophilic peptide sequences incorporated into lipid bilayers perturb the packing of lipids and affect their thermotropic properties, more than the non-structural selected sequence. However, the binding of the synthetic sequences to lipid membranes occurred without any restructuration of the peptides.

Differences in secondary structure of HAV-synthetic peptides induced by the sequential order of T- and B-cell epitopes.

The present study was undertaken to examine the structural features of two peptide constructs designed on the basis of linear combination of B and T-cell epitopes in different orientations (BT and TB) that may be important to explain the differences in the elicited antihepatitis A virus immune response and in the interaction with biological model membranes. A CD study was carried out and the corresponding quantitative analysis of the experimental data was done using deconvolution computer programs. Moreover, fluorescence experiments were performed to analyze differences in the fluorescence emission spectra of both molecules. The main conformational difference by CD studies was obtained working in aqueous medium. Although the TB sequence adopted a preferably random coil structure, the BT peptide was best fitted with beta-type structures. These results are further supported by fluorescence studies. These findings have relevance for the design of synthetic immunopeptides.

Assessment of synthetic peptides for hepatitis A diagnosis using biosensor technology.

In the present work we demonstrate the application of a commercial biosensor instrument (BIACORE 1000, Biacore AB, Uppsala) for the detection of antibodies against the hepatitis A virus (HAV) in human serum samples using linear and branched synthetic peptides related to the VP3 capsid protein of HAV. We also studied the conformation of the synthetic peptides by circular dichroism (CD) in order to analyse the changes in secondary structure of the constructs that could influence their recognition by antibodies. Linear and dimeric VP3(110-121) multiple antigen peptides (MAP) were the most sensitive and appropriate for serological studies of serum from HAV infected patients using BIACORE. Immobilization of tetrameric MAPs via amine groups apparently failed to preserve the active conformation of the peptide epitope since it led to lower antibody binding compared to linear and dimeric peptides. The CD analysis showed that the tetrameric MAP constructs tend to adopt a beta-sheet structure due to intermolecular aggregation, which limits epitope accessibility. Our results demonstrate the value of biospecific interaction analysis technology using synthetic peptides for the diagnosis of acute hepatitis A.

Use of linear and multiple antigenic peptides in the immunodiagnosis of acute hepatitis A virus infection.

The reactivities of two panels of anti-HAV human sera from geographically distinct areas (Chile and Spain) to synthetic peptides from the VP1, VP2 and VP3 hepatitis A virus capsid proteins were examined by an enzyme-linked immunosorbent assay (ELISA) procedure. Two and four branched multiple antigenic peptides (MAPs) and palmitoylated peptides were compared with free synthetic sequences for the detection of IgM anti-HAV antibodies in the two panels of human sera. Our results showed that acute hepatitis A patient sera recognized preferentially homogeneous two branched MAPs and palmitic acid conjugated peptides. The palmitoyl-derived VP3(110-121) peptide and the corresponding dimeric MAP were the most sensitive and appropriate for serological studies of HAV-infected patients by ELISA, sensitivity and specificity being higher than 90% and 95%, respectively. These peptide-based tests open up new avenues in the development of peptide-based immunosorbent assays for the detection of acute HAV disease.

Different approaches to potentiate the immune response induced by a 12-mer synthetic peptide.

Interest in synthetic immunogenic peptides is increasingly growing due to the continuous achievements in the understanding of the molecular basis of the immune response. Moreover, the use of peptide fragments to generate antibodies capable of recognizing and neutralizing viral particles could be a valuable alternative to conventional vaccines: they are safe, they can be designed to induce defined immune responses and they can be synthesised in large quantities in high purity. However, their relatively low immunogenicity requires the development of effective adjuvants and/or their incorporation into controlled release formulations. Several different strategies have been used for the induction and analysis of protective immune responses induced by short peptides against infectious diseases. In the present article we summarise the different approaches used to potentiate the immune response induced by a continuous epitope of hepatitis A virus: co-linear link of T-cell epitopes in different orientations, incorporation into liposomes, and preparation of homogeneous and heterogeneous Multiple Antigenic Peptides.

Enhancement of the immunogenicity of a synthetic peptide bearing a VP3 epitope of hepatitis A virus.

The immune responses elicited in mice by different forms of the VP3(110-121) B-epitope of the hepatitis A virus (HAV) were studied. Different forms of incorporation in liposomes were tested, encapsulation, rather than surface exposure, being the best antigenic preparation. Three larger peptides of the VP3 epitope, two of them containing a hepatitis B virus T-epitope, and a third containing a putative T-epitope of HAV (VP3(102-121)) were assayed. While this latter T-epitope induced an enhancement of the response against the VP3 B-epitope, the artificially coupled T-epitopes failed to induce a significant increase. The administration of two multiple antigenic peptide (MAP) constructs, the first containing the VP3(110-121) and VP1(11-25) HAV sequences and the second only the VP1(11-25) sequence, also suggested the presence of a T-epitope, since the response against the VP1 peptide was increased in the first construct.