María J. Gómara

Effect of Synthetic Peptides Belonging to E2 Envelope Protein of GB Virus C on Human Immunodeficiency Virus Type 1 Infection

The use of synthetic peptides as HIV-1 inhibitors has been subject to research over recent years. Although the initial therapeutic attempts focused on HIV-coded enzymes, structural HIV proteins and, more specifically, the mechanisms that the virus uses to infect and replicate are now also considered therapeutic targets. The interest for viral fusion and entry inhibitors is growing significantly, given that they are applicable in combined therapies or when resistance to other antiretroviral drugs is seen and that they act before the virus enters the cell. The 124 synthetic sequences of the GBV-C E2 envelope protein have been obtained by SPPS. The interaction of certain GBV-C peptide sequences with the HIV-1 fusion peptide has been proven through the use of biophysical techniques. We also show how GBV-C E2 domains notably decrease cellular membrane fusion and interfere with the HIV-1 infectivity in a dose-dependent manner, highlighting their potential utility in future anti-HIV-1 therapies.

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.

Diagnostic and prognostic value of antibodies against chimeric fibrin/filaggrin citrullinated synthetic peptides in rheumatoid arthritis

IntroductionEvidence suggests that citrullinated fibrin(ogen) may be a potential in vivo target of anticitrullinated protein/peptide antibodies (ACPA) in rheumatoid arthritis (RA). We compared the diagnostic yield of three enzyme-linked immunosorbent assay (ELISA) tests by using chimeric fibrin/filaggrin citrullinated synthetic peptides (CFFCP1, CFFCP2, CFFCP3) with a commercial CCP2-based test in RA and analyzed their prognostic values in early RA.MethodsSamples from 307 blood donors and patients with RA (322), psoriatic arthritis (133), systemic lupus erythematosus (119), and hepatitis C infection (84) were assayed by using CFFCP- and CCP2-based tests. Autoantibodies also were analyzed at baseline and during a 2-year follow-up in 98 early RA patients to determine their prognostic value.ResultsWith cutoffs giving 98% specificity for RA versus blood donors, the sensitivity was 72.1% for CFFCP1, 78.0% for CFFCP2, 71.4% for CFFCP3, and 73.9% for CCP2, with positive predictive values greater than 97% in all cases. CFFCP sensitivity in RA increased to 80.4% without losing specificity when positivity was considered as any positive anti-CFFCP status. Specificity of the three CFFCP tests versus other rheumatic populations was high (> 90%) and similar to those for the CCP2. In early RA, CFFCP1 best identified patients with a poor radiographic outcome. Radiographic progression was faster in the small subgroup of CCP2-negative and CFFCP1-positive patients than in those negative for both autoantibodies. CFFCP antibodies decreased after 1 year, but without any correlation with changes in disease activity.ConclusionsCFFCP-based assays are highly sensitive and specific for RA. Early RA patients with anti-CFFCP1 antibodies, including CCP2-negative patients, show greater radiographic progression.

Conjugation of cell-penetrating peptides with poly(lactic-co-glycolic acid)-polyethylene glycol nanoparticles improves ocular drug delivery

In this work, a peptide for ocular delivery (POD) and human immunodeficiency virus transactivator were conjugated with biodegradable poly(lactic-co-glycolic acid) (PGLA)–polyethylene glycol (PEG)-nanoparticles (NPs) in an attempt to improve ocular drug bioavailability. The NPs were prepared by the solvent displacement method following two different pathways. One involved preparation of PLGA NPs followed by PEG and peptide conjugation (PLGA-NPs-PEG-peptide); the other involved self-assembly of PLGA-PEG and the PLGA-PEG-peptide copolymer followed by NP formulation. The conjugation of the PEG and the peptide was confirmed by a colorimetric test and proton nuclear magnetic resonance spectroscopy. Flurbiprofen was used as an example of an anti-inflammatory drug. The physicochemical properties of the resulting NPs (morphology, in vitro release, cell viability, and ocular tolerance) were studied. In vivo anti-inflammatory efficacy was assessed in rabbit eyes after topical instillation of sodium arachidonate. Of the formulations developed, the PLGA-PEG-POD NPs were the smaller particles and exhibited greater entrapment efficiency and more sustained release. The positive charge on the surface of these NPs, due to the conjugation with the positively charged peptide, facilitated penetration into the corneal epithelium, resulting in more effective prevention of ocular inflammation. The in vitro toxicity of the NPs developed was very low; no ocular irritation in vitro (hen’s egg test–chorioallantoic membrane assay) or in vivo (Draize test) was detected. Taken together, these data demonstrate that PLGA-PEG-POD NPs are promising vehicles for ocular drug delivery.

Anti-citrullinated peptide antibodies in the serum of heavy smokers without rheumatoid arthritis. A differential effect of chronic obstructive pulmonary disease?

The objective of this study is to analyse the frequency and levels of anti-citrullinated peptide/protein antibodies (ACPA) in the serum of non-rheumatoid arthritis (RA) heavy smokers with and without chronic obstructive pulmonary disease (COPD) and compare them with healthy never smokers and patients with RA. Serum samples of 110 heavy smokers without RA, 209 healthy never smokers and 134 patients with RA were tested for ACPA using a commercial anti-cyclic citrullinated peptide antibodies (CCP2) test and a homemade chimeric fibrin/filaggrin citrullinated synthetic peptide (anti-CFFCP) ELISA test. The frequency of positive results and autoantibody levels were compared between groups. The prevalence of the two types of ACPA was slightly higher in heavy smokers than in never smokers, although the difference was not significant, and significantly lower than in RA patients. The highest prevalence of positive ACPA in heavy smokers was found in subjects with COPD (7.4% of positive anti-CFFCP in patients with COPD in comparison with 2.4% in never smokers: OR 3.26; 95% CI 0.85–12.6, p = 0.089). Mean serum levels of ACPA in heavy smokers were not significantly different from those of never smokers. Heavy smokers with COPD had significantly higher levels of anti-CFFCP than those without COPD, although almost all patients had serum levels below the cut-off values. The prevalence of ACPA in heavy smokers without RA is low, but seems to be higher in heavy smokers with COPD. Larger studies are necessary to confirm these findings and determine the relationship between ACPA and lung disease.

Assessment of synthetic chimeric multiple antigenic peptides for diagnosis of GB virus C infection

The use of synthetic peptides of both structural and nonstructural proteins of GB virus C (GBV-C) has been studied for the development of new systems to diagnose infection caused by this virus. In an attempt to increase the antigenicity of linear peptide sequences, chimeric multiple antigenic peptides (MAPs) containing epitopes from E2, NS4, and NS5 GBV-C proteins have been synthesized. The synthetic constructs were evaluated by ELISA to establish whether the epitopes in chimeric branched peptides are more efficiently recognized by the specific antibodies compared to the monomeric linear sequences. Moreover, we have investigated the application of a commercial biosensor instrument for the detection of antibodies against the GBV-C in human serum samples. The results of the immunoassays reported in this work highlight the usefulness of synthetic tetrameric branched peptides containing sequences from envelope and nonstructural GBV-C proteins for the diagnosis of GBV-C infection. The potential clinical value of the MAP(4)(E2-NS5a) for the serodiagnosis of GBV-C infection was demonstrated, thus providing the basis for performing prevalence studies of the infection among the hemodialyzed and hepatitis C virus (HCV)-infected population.

Design of synthetic peptidic constructs for the vaccine development against viral infections.

The practical development of modern vaccines has been greatly advanced by the availability of synthetic antigens. The use of such synthetic antigens might be more acceptable for human therapy since synthetic peptides do not have any of the potential dangers associated with the induction of an infection by recombinant viruses. However, synthetic peptides alone are often not immunogenic enough, and a strong immunoadjuvant is usually employed for their elaboration. Unfortunately, only a few adjuvants used in experimental models are allowed for use in human beings. In this regard, different presentations of synthetic peptides such as incorporation into liposomes, modification of the lipophilic properties by means of a covalently coupled fatty acid moiety and the synthesis of larger constructs such as multiple antigenic peptides (MAP) have been demonstrated to yield efficient immunological reagents for the amplification in the analysis and induction of immune responses to a variety of infectious agents. This review outlines recent research on synthetic peptide immunology. The development of a MAP with a built-in adjuvant is highlighted as a robust method for vaccine design.

Analysis of HIV-1 fusion peptide inhibition by synthetic peptides from E1 protein of GB virus C

The aim of this study was to identify proteins that could inhibit the activity of the peptide sequence representing the N-terminal of the surface protein gp41 of HIV, corresponding to the fusion peptide of the virus (HIV-1 FP). To do this we synthesized and studied 58 peptides corresponding to the envelope protein E1 of the hepatitis G virus (GBV-C). Five of the E1 synthetic peptides: NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18) and AQLVGELGSLYGPLSVSA (P22) were capable of inhibiting the leakage of vesicular contents caused by HIV-1 FP. A series of experiments were carried out to determine how these E1 peptides interact with HIV-1 FP. Our studies analyzed the interactions with and without the presence of lipid membranes. Isothermal titration calorimetry revealed that the binding of P7, P18 and P22 peptides to HIV-1 FP is strongly endothermic, and that binding is entropy-driven. Gibbs energy for the process indicates a spontaneous binding between E1 peptides and HIV-1 FP. Moreover, confocal microscopy of Giant Unilamellar Vesicles revealed that the disruption of the lipid bilayer by HIV-1 FP alone was inhibited by the presence of any of the five selected peptides. Our results highlight that these E1 synthetic peptides could be involved in preventing the entry of HIV-1 by binding to the HIV-1 FP. Therefore, the continued study into the interaction between GBV-C peptides and HIV-1 FP could lead to the development of new therapeutic agents for the treatment of AIDS.

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.

Diagnostic Value of Anti-GBV-C Antibodies in HIV-Infected Patients

The beneficial effect of co‐infection by GB virus C (GBV‐C) in the course of the disease in human immunodeficiency virus (HIV)‐infected patients has been described, although its mechanism of action is yet to be determined. The role of anti‐GBV‐C antibodies in HIV‐infected patients also remains unknown. At present, there are no commercial systems to detect specific markers of GBV‐C infection. The research presented follows our previous work from which we obtained chimeric molecules formed by two domains of different GBV‐C proteins with good sensitivity/specificity balances in the detection of anti‐GBV‐C antibodies in hemodialyzed and chronic hepatitis patient samples. It has been investigated the ability of the synthetic peptides to recognize specific anti‐GBV‐C antibodies in HIV and HCV/HIV co‐infected patients by a peptide‐based ELISA immunoassay. The results showed that human immunodeficiency virus‐infected patients have a significantly higher frequency of anti‐GBV‐C antibodies than healthy controls. A comparison between HCV+/HIV+ and HCV−/HIV+ was analyzed. Although a higher percentage of HCV/HIV‐positive sera were positive for antibodies against GBV‐C peptides, the difference was not significant. The presence of anti‐GBV‐C antibodies could represent a good marker of exposure to GBV‐C in HIV‐infected patients to facilitate a further analysis of the effect of this exposure in the progression of illness caused by HIV infection.