At the beginning of this research program, we observed a direct correspondence between the most hydrophilic regions of a protein [29] and its antigenicity [30]. The systematic use of computer algorithms selects more efficiently the regions of a protein that are potentially the most antigenic. This saves time and reagents and leads to the reduction of experimental animals, among other factors.

In general, there are three basic important requirements in the selection of B-cell epitopes: hydrophilicity, flexibility, and accessibility or exposure for interaction with antibodies, which allow the selection of exposed, mobile, and hydrophilic regions so that antibodies can bind to them effectively and generate an immune response. Incorporation of programs (InsightII^®) allowed us to observe the 3D structure and relate the antigenicity observed in certain synthesized peptides with the topological location in the respective protein.

Exposed regions are those that interact more with the environment that surrounds the protein (rather than what is inside the protein structure). Those very flexible regions form secondary structures called “random arrangements” and “turns”. They are generally more antigenic [31], unlike structures such as alpha helices or beta sheets, dominated by van der Waals-type forces and hydrogen or disulfide bonds that make them more rigid. The amino and carboxy-terminal regions of a protein generally meet these requirements, too.

Prediction programs were replaced with free access online programs that can be read on personal computers with internet access. Several algorithms have been developed in the last decades, and with their incorporation into online web portals, they have become more efficient and complex. Other physicochemical characteristics (many derived from experimental data) were added. Some of the most frequently used algorithms by our research group are listed in Table 1.

The arsenal of immunoinformatic algorithms was enhanced by the availability of crystallized structures [X-ray or nuclear magnetic resonance (NMR)] extracted from the Protein Data Bank (PDB) (https://www.rcsb.org).

In a similar approach but looking for Th1-type T-cell epitopes, immunoinformatics studies have been carried out. Up to six immunoinformatics algorithms were used and listed in Table 2.

Artificial intelligence (AI), which is acquiring increasing relevance, has recently been introduced and used in predictive studies of 3D structures of proteins not yet crystallized. It is a great tool for the visualization and selection of epitopic regions [32].

Synthetic peptides have won notoriety thanks to developments in ​​biotechnology and are currently a key component of nanotechnological applications. Their chemistry is an area of ​​research with many approaches, from vaccine design, drug development, drug delivery systems, and antimicrobial peptides to diagnostic methods and disease treatment. The peptides are very useful for the detection of various human and veterinary pathologies [33–38].

The design of peptide libraries and their subsequent evaluation by immunoenzymatic methods permits the rapid screening of sequences of interest, considerably reducing the cost of synthesis per peptide compared to its conventional synthesis on polymer resins. Such is the case of the SPOT technique, which is easily integrated into research laboratories manually or semi-automatically, allowing the quantity of synthesized peptides to be varied. It can even integrate modifications, in addition to sieving or screening the peptide library in matrix form, which is widely used for the detection of peptide-ligand interactions [39, 40].

Bioinformatics tools, the SPOT, and their evaluation with immunoglobulins IgG, IgM, and IgA through the PEPSCAN immunoassay of structural and non-structural proteins were combined to identify potential candidate antigenic peptides to determine possible biomarkers of infection [41].

Our aim was to use this strategy to develop a commercial kit for the simultaneous and multiple diagnosis of endemic diseases with the appropriate mimicking of the antigenic proteins. Figure 3 describes the strategy we are currently using for the identification of antigenic peptides as candidates for diagnosis. These general steps are usually cyclical, and their number of repetitions is subject to the results obtained during the process. Optimizing the most antigenic peptides is possible by carrying out a series of structural modifications, such as: the design and synthesis of MAPs [12], site-directed mutagenesis by alanine scanning for the identification of critical residues [42], and obtaining 3D models of the synthesized peptides using PEP-FOLD [43] and its structural analysis.

For the immunological evaluation, first, SPOT synthesis was performed with peptides usually having a 16–20 AA length. Peptides were constructed on a gridded 15 × 15 cm cellulose filter paper (Whatman 50 or 540), and each spot was numbered using a graphite pencil. The membrane was exposed to a β-alanine solution, then deprotected with 20% 4-methylpiperidine/DMF (dimethylformamide) or NMP (N-methyl pyrrolidone) with acetic anhydride/diisopropyl ethylamine.

Afterward, it was stained with bromophenol blue (BPB), at which point the entire membrane should turn blue. Between 0.5 and 1.0 μL of the first AA, derived from the respective peptide sequence, was dissolved in DMF or NMP and previously activated. Next, the AA was coupled on each numbered spot, allowing it to react for 20 min, then this step was repeated. The membrane was acetylated using a 2% acetic anhydride/diisopropylamine mixture and stained with BPB. Finally, the Fmoc group on each spot was removed by subjecting it to a 20% 4-methylpiperidine solution for 5 min twice. After washing with NMP and methanol and subsequent staining with BPB, each spot should be colored blue.

The coupling, acetylation, and deprotection steps were repeated for each AA of the respective peptide sequence of each SPOT until the desired peptides were obtained. Finally, these were deprotected in their side chains using a mixture of 95% trifluoroacetic acid, 2.5% water, and 2.5% triisopropyl amine [44].

For the immunological evaluation, the membrane with the peptides to be evaluated was blocked with 5% skimmed milk in phosphate buffer saline with 0.05%-Tween 20 (PBST). After successive washes with PBST, it was exposed to a pool of serum dissolved in a 1/200 ratio for 90 min, subsequently incubated in an anti-antibody-enzyme conjugate solution (IgG, IgM, IgA, or IgE) overnight, and then a chemiluminescent or colorimetric substrate was added. Positive peptides were observed as spots in different shades of gray or colored if a colorimetric substrate was used. These positive peptides were then produced in large quantities and used for evaluation using ELISA and/or MABA.

Conventional ELISAs are made by coating the plates with a 2–5 µg/mL dilution of the peptides, 1:100 dilution of sera, and using p-nitrophenylphosphate (p-NPP) as substrate, as previously described [11].

MABA is a dot-blot assay in which different antigens are adsorbed onto nitrocellulose paper, and 0.45 µm is inserted into a commercial or home-made acrylic chamber with 28 channels in carbonate bicarbonate buffer (pH 9.6) for 60 min [3]. Each strip can adhere up to 26 different antigens and two positive controls.

In our experiment, normal human serum (HS) was used as a source of immunoglobulins for positive controls. The nitrocellulose membrane was blocked with skimmed milk (5%) in PBST and cut perpendicularly to allow the 28 adsorbed antigens to be simultaneously exposed to the diluted body fluid (1:100 in blocking buffer). Secondary antibodies with enzymes were used to reveal positive signals with the corresponding substrates and were exposed to patient sera for antibody detection. Anti-antibodies conjugated to enzymes showed positive reactions as small colored boxes.

Manually-synthesized peptides derived from conserved regions of HIV-1 glycoproteins 41 and 120 (gp41, gp120) were designed. The gp41 protein is a surface protein of HIV, and antibodies to the gp41 protein can be used to diagnose HIV infection [58]. The gp41 protein is a candidate for HIV-1 diagnosis; it has a high reactivity with HIV-1-positive sera and, therefore, a high antigenicity. Three pairs of synthetic peptides, 2 pairs derived from the HIV-1 gp41 and one from the gp120, were tested with HIV sera using MABA. Each pair consists of the same peptide, with some modifications. IMT-1954 is a 19-AA sequence derived from the gp41 protein that contains CG in the N-terminal and glycine-cysteine (GC) in the C-terminal. The addition of CG and GC AAs favors its polymerization and yields a higher reactivity by increasing the number of binding sites to patient antibodies [59]. We have observed in previous works carried out in our laboratory that polymeric peptides are more antigenic and immunogenic.

The peptide IMT-2197 is a modification of the p41 IMT-1954 with CG only in the N-terminal, allowing the formation of dimers. The peptide IMT-1961 is derived from the same protein (gp41) but contains part of the peptides IMT-1954 and IMT-2197 as a dimer. Peptides IMT-1965 (dimer) and IMT-1968 (polymer) were derived from the HIV-1 gp120 sequence. Different degrees of reactivity to these peptides were found in the HIV serum samples. In our studies, 146 sera [108 with positive polymerase chain reaction (PCR) results] were tested with a recognition frequency of 86.6% for the peptide IMT-1954 (non-published data, Figure 4, Table 3). The peptide IMT-2197 showed a recognition frequency of 82.8% in 105 patients. With the mixture of peptides IMT-1954 + 1961 + 2197, the recognition was 100% in 41 PCR-positive patients. Using MAPs from different proteins guarantees maximum recognition and avoids the possibility of a lack of response due to genetic background. In previous synthesis processes, these peptides were evaluated with 37 HIV-positive sera and were recognized by 86.5–91.9% of those patients. On the other hand, comparing patients with or without treatment, each of the peptides IMT-1954 and 2197 had a recognition frequency of 100% in 11 untreated patients, which decreased to 86.6% in 15 patients after treatment (Figure 5). Specificity was always 100%; this means there was no cross-reaction, and all negative sera evaluated were negative for this test. These peptides could be used for follow-up during antiretroviral therapy since it is known that patients under treatment exhibit attenuated responses, a phenomenon also reflected in rapid tests.

Our team, when working as Durango et al. [46], aimed to analyze the immune response to HIV synthetic peptides in different ethnic groups from Venezuela, with a particular interest in the indigenous Warao population, facing a devastating HIV-1 epidemic. The Warao displayed higher frequencies of recognition for all peptides. This distinctive pattern suggests a unique immune response profile within the Warao group. The antibody response in this group was notably robust, contrary to our expectations, considering their rapid evolution to acquired immunodeficiency syndrome (AIDS). This group revealed a surprisingly resilient antibody response, challenging prevailing notions regarding immune responses in unique subpopulations and highlighting the importance of considering population-specific factors in HIV research.

Our peptides detected HIV-1-positive samples in the general Venezuelan population and in an indigenous Warao population from Venezuela. Specificity was 100% since no reactivity was observed with negative sera. All of the peptides were recognized by three sera from HIV-1-positive samples infected with subtype A1. HIV-1 patients under antiretroviral therapy exhibited a lower frequency and intensity of peptide recognition.

Our study revealed that there was a significant difference in the antibody reactivity pattern between samples from patients with antiretroviral therapy and those from the Warao population for almost all of the peptides. The Warao group recognized the peptides with higher intensity than other ethnic groups. The application of MABA demonstrated its efficacy in detecting antibody reactivity patterns among different cohorts. This technique effectively identified specific peptides that were particularly recognized, showcasing its potential for analyzing the antibody response in depth [2]. The use of synthetic peptides, coupled with the robust performance of MABA, enriches our understanding of antibody responses in different HIV-1-infected populations.

Synthetic peptides have also proven to be useful tools for the immunodiagnosis of hepatitis C virus (HCV) infection. Synthetic peptides (monomers) from the subtype 1a polyprotein from the HCV core (40-AA IMT-286 N-terminal and 20-AA 716 derived from a region located inside 286), NS4 (20-AA IMT-59 that contains a highly conserved sequence among different HCV isolates), and NS5 (35-AA IMT-290) regions were evaluated using ELISA. The mixture of peptides IMT-286, -59, and -290 detected antibodies in 100% of the immunocompetent infected patients and in 91% of the immunocompromised patients. Therefore, this peptide cocktail seems to be useful for epidemiological studies in the general population [47].

Thereafter, these peptides were used to diagnose the hepatitis C seroprevalence in Venezuela [48]. A total of 2,592 sera from urban, rural, and indigenous populations were tested with an in-house-developed immunoassay based on the previously mentioned synthetic peptides. In summary, 39 of the samples were found to be positive for HCV peptides. This in-house, synthetic-peptide-based immunoassay seems to be a valuable tool for epidemiological studies. To improve previous results and to decrease the size of one of the previous reactive peptides of 40-AA, we reanalyzed the core, E2, NS3, NS5A, structural, and non-structural viral proteins using immunoinformatics to select other antigenic peptides that were tested initially with the PEPSCAN technique [49]. Next, we performed the immunodiagnosis of HCV infection using MABA with modifications of these peptides with and without stearic acid at the N-terminal. A 20-AA linear peptide (IMT-2052) belonging to the N-terminal of the worldwide conserved core protein without the addition of stearic acid, showed 100% sensitivity and specificity in 22 confirmed patients [49]. This strategy (PEPSCAN plus MABA) allowed the selection of this peptide sequence with just 20 residues from the very conserved core protein, which leads to a low-cost alternative for prevalence studies and early diagnosis of HCV infection and highlights its relevance in the immunodiagnosis of viral hepatitis C.

A group of antigens from the exposed capsid proteins VP1, VP2, and VP3 that were previously known for their antigenicity was selected [50]. After its synthesis and the corresponding reactivity analysis by PEPSCAN using a pool of sera from hepatitis A virus (HAV) patients, a 15-AA peptide (IMT-1996) was selected for greater-scale synthesis on resin, one of which was derived from the C-terminal of the VP1 protein. The peptide was then evaluated using MABA and ELISA in a dimeric form, with a sensitivity of 87–100% with anti-IgG antibodies and 100% with anti-IgM. The HAV infection is usually an acute disease, in contrast with hepatitis B virus and HCV. Therefore, as shown in our assays and previously expected, the diagnosis is more sensitive in detecting IgM. The C-terminal region of the protein VP1 has been previously recognized as antigenic by others [60–63]. This recombinant antigen has been used successfully in epidemiologic studies [64–66].

However, synthetic peptides are easier to produce, more chemically stable, and less expensive than recombinant molecules, especially with sequences shorter than 20 residues. The evaluation of this peptide using saliva in addition to sera and IgA as immunoglobulin is ongoing.

We have also obtained good results using synthetic peptides derived from parasitic agents, such as Trypanosoma cruzi (T. cruzi), the pathogen responsible for Chagas disease. The reactivity of chagasic sera against a group of peptides covering most of the heat-shock protein Hsp70 has shown that > 50% of the peptides had a positive response, but a high reactivity was observed only against a few peptides (9, 12, 14, and 37) in 9 positive sera [51]. The general pattern of reactivity against the peptides is different in chagasic and healthy sera.

For the discrimination of the clinical stages of Chagas disease, we described those three out of five different repeats contained in the T. cruzi TcCA-2 membrane protein (3972, 6303, and 3973) that are recognized by > 90% of chronic Chagas disease patients but not by patients in the acute phase of the disease [52]. The 3973 peptide shows a specificity of > 98% since it is not recognized by patients with autoimmune and inflammatory diseases, nor by patients with a non-chagasic cardiomyopathy. It is noteworthy that the antibody levels against the 3973 epitope detected by ELISA with the sera from Chagas patients in the symptomatic chronic phase, involving cardiac or digestive alterations, are higher than those detected by the sera from Chagas patients in the indeterminate phase of the disease. We suggest that this diagnostic technique could also be used as a biomarker of pathology. We determined that certain AAs [F, Q, and diketopiperazine (DKP), located in the peptide at positions 1, 3, and 8 to 10, respectively] are essential to the conformation of the immunodominant antigenic epitope.

Malaria is still a serious public health problem across the world, affecting hundreds of thousands of people annually, mainly in tropical countries. That is why it is crucial to make an accurate diagnosis in order to apply the appropriate treatment. Derived from Plasmodium falciparum (P. falciparum) excretory-secretory proteins {the glutamate-rich protein (GLURP), the histidine-rich protein 2 (HRP2), the HRP3 [the falciparum interspersed repeat antigen (FIRA)], and the serine-rich antigen homologous P. falciparum (SERPH)}, peptides were designed to be used in an antigen-capture assay [27]. Conformational and antigenic predictors were used. Polyclonal antibodies in rabbits were prepared using the peptides as antigens. Eight out of 14 constructs by ELISA and six by MABA elicited antibodies, corresponding to the predictive study and the immunogenicity obtained in rabbits. Western blots were performed with P. falciparum cultures to determine the binding activity and specificity of the anti-peptide antibodies, reacting against five molecules from 73 to 128 kDa. An inhibition assay was induced by preincubation with the homologous peptides.

Interestingly, the antibodies anti-HRP2 recognized the recombinant protein included in the ParaSight-F rapid test. In the same way, synthetic peptides from the HRP2 molecule were recognized by the monoclonal antibody present in the same rapid test. This confirms the potential value of synthetic peptides to induce monospecific polyclonal antibodies for developing antigen-capture assays.

Attempts to develop antigen-capture assays in blood and saliva have been performed. These peptides from the repetitive region of the GLURP, previously identified as IMT-94A, -154, and -200, and the peptide IMT-192 of HRP2 [27] were exposed to malaria patients’ sera and saliva in PEPSCAN and were recognized with frequencies up to 47% in sera only, not in saliva (data non published). Preliminary results have been obtained with a group of peptides: IMT-2079 and -2082 (RAP-3), -2087 (MSP-9), and -2089 (FIRA), that were used to immunize hens in order to obtain specific IgY antibodies from the eggs. IgY against peptides IMT-2087 and -2089 were successfully produced by the hens, and now we are in the process of evaluating if it is possible to detect these antigens in a capture assay.

Extensive studies have been developed on the diagnosis of schistosomiasis since the active search for potentially infected communities requires massive, sensitive diagnosis to detect asymptomatic cases of very low parasitic load [53–55]. Antigenic peptides derived from the proteins Sm31 (cathepsin B) and Sm32 (asparaginyl endopeptidase) were evaluated using 51 human sera infected with Schistosoma mansoni (S. mansoni) [53]. Their immunogenicity in rabbits (in order to produce monospecific polyclonal antibodies) was measured using MABA [54]. Peptides IMT-164 and -180 from Sm31, when combined, showed sensitivities up to 96% with 100% specificity. Peptides from Sm31 were evaluated for antigenicity (in humans) and immunogenicity (in rabbits). The individual sensitivities ranged between 22.5 and 67.5%. Antigen-capture assays developed from rabbits immunized with synthetic peptides 487, 488, 489, and 493 recognized the native antigen in 62.5% of the patients’ sera [55].

This is of particular relevance in areas of low and moderate intensities of infection (as low as 24 eggs/g feces), together with the evaluation of the efficacy of chemotherapy. With the same approach, 20-AA long peptides comprising the entire Sm31 molecule of the adult worm were synthesized, and their immunogenicity was evaluated in rabbits [56]. Peptides IMT-172 and -180 induced antibodies that adhered to the adult worm gut, where the Sm31 is present. They were also responsible for the recognition of the native Sm31 molecule via WB and by immunocytochemistry of cross-sections of adult worms [30]. Twenty-two polymeric peptides derived from Sm32 were synthesized, and rabbits were immunized [28]. Seventeen peptides were found to be immunogenic, and sera from immunized rabbits corresponding to the molecule from the first 335 AAs recognized the 32 kDa native protein from the adult worm antigen via WB. Sm32 is one of the excretory-secretory molecules released with the vomitus of the adult worm and is one of the target antigens for detection in the plasma of infected individuals. Sm32 was also studied thoroughly using “in silico” modeling and structural analysis [67] to assess its immunological and drug target implications. Sm32 can be exploited as a potential target for the development of biomarkers used in diagnosis.

Synthetic peptides are useful in studying the phylogenetic relationship between different schistosome species and also for application in diagnosis (pan-diagnosis) and/or cross-protective vaccines [68].

RT PCR (real-time PCR) and antigen-detection tests are more useful for diagnosing acute COVID-19, while antibody tests measure the population’s exposure. There is an urgent need for the development of diagnostic tests that can also be used for large-scale epidemiological screening, immunological screening, and the coverage of vaccine programs. A detailed analysis of the three structural proteins [spike (S), nucleocapsid (N), and membrane (M)] and the non-structural proteins [open reading frames (ORFs)] present in the SARS-CoV-2 virus was conducted by combining immunoinformatic methods, peptide SPOT synthesis, and the PEPSCAN immunoassay [44]. Sera from patients’ pools in PEPSCAN showed a strong signal for peptides corresponding to the S, N, and M proteins of the virus, but not for the ORFs. The peptides exhibiting higher signal intensity were found in the C-terminal region of the N protein, showing different levels of recognition with IgM, IgG, and IgA isotypes. This difference in reactivities can ensure accurate diagnosis of infected patients, including their infection exposure times. A validation in the general population using an ELISA in-house assay is being performed to determine the seroprevalence of SARS-CoV-2/COVID-19 in Venezuela.

In a subsequent investigation, using immunoinformatic tools, continuous and discontinuous linear B-cell epitopes were identified for the N and M proteins of the SARS-CoV-2 virus. Peptides were synthesized using the SPOT method, and their antigenicity was evaluated using PEPSCAN with groups of sera for three immunoglobulins (IgG, IgM, and IgA). The prediction of the secondary structure of these sequences was carried out using PEP-FOLD, and the secondary structure obtained was compared with the corresponding region of the crystallized M protein. These were synthesized in solid-phase on resin and evaluated using the ELISA immunoenzymatic technique to determine their antigenicity, obtaining sequences that proved to be the most antigenic. These studies are still in progress.

A subsequent screening allowed us to identify sequences that showed the highest percentages of sensitivity and specificity. Individual peptides and mixtures were evaluated in ELISA to determine interactions that increase the sensitivity of the assay, showing in some cases an increase in sensitivity and specificity when mixtures were used to compare with individual peptides (results not yet published). This research allowed us to obtain antigenic peptides from the N and M proteins that can be used for the development of methods for diagnosing and monitoring COVID-19.

The methodology employed by our research group for the identification of B-cell epitopes has also been successfully applied in the identification of potential T-cell epitopes, applying each of the above-mentioned algorithms to the selected COVID-19 proteins [S, receptor binding domain (RBD), N, and M]. The best epitopes are being evaluated by measuring the release of interferon gamma by blood cells in patients with COVID-19 exposed to peptides [69].

We also developed valuable synthetic peptides for the diagnosis of the gram-negative bacteria, H. pylori, which colonizes the human gastric mucosa. Its role in the pathogenesis of gastritis, peptic ulcer disease, and gastric cancer has been demonstrated, where the protein from H. pylori (CagA) has an important role in the development of gastric carcinogenesis [70, 71].

The effector protein CagA is expressed by the highly virulent H. pylori strains possessing cagPAI, and it is absent from less virulent cagPAI-negative strains. The biological activity of CagA is determined by the number of repeats of the sequence glutamic acid-proline-isoleucine-tyrosine-alanine (EPIYA), present in the C-terminal region.

The diagnosis of this infection is complex, mainly due to the inconsistency that may exist between the available diagnostic tests since few tests detect its virulence factors [72]. The diagnosis of strains that produce the oncogenic protein CagA contributes to evaluating the risk of serious gastric diseases, guiding treatment, and monitoring infection. In this context, various sequences of the CagA were analyzed according to the methodology previously described, and different peptides were synthesized, obtaining 11 peptides of high sensitivity and specificity when determining IgG and secretory IgA in 349 sera and 390 saliva from infected individuals evaluated in Venezuela, both by MABA (Figure 6). A moderate level of agreement (Cohen’s kappa = 0.58) was observed when comparing these results with the detection of gene from H. pylori (cagA)-positive strains in patient biopsies using PCR.

Moreover, an ELISA assay was standardized, and six peptides from the CagA were selected based on their previously demonstrated good reactivity and sensitivity as determined by MABA (IMT-1436, -1440, -1441, -1442, -1444, and -1445), as well as a pool of these peptides (CagA pool). Peptides 1442, 1444, and 1445, which showed the highest antigenicity, were selected, and secretory IgA and serum IgG antibodies were determined in samples from patients positive and negative for the cagA detected by PCR. Statistically significant differences were observed in the median values of secretory IgA and serum IgG antibodies between patients with the cagA compared to those without the cagA, and these in turn were compared with uninfected control patients. It should be noted that the studies have been carried out in symptomatic patients who, although negative at the time of evaluation, may have had a previous infection, so they may have specific memory antibodies.

Furthermore, the results demonstrate that secretory IgA had the highest recognition for all peptides, including the pool, compared to IgG. Some peptides were able to induce a stronger secretory IgA response (in saliva) than IgG (in serum), suggesting that these two types of antibodies recognize distinct epitopes in the peptides, reflecting the differences between the systemic IgG response and the mucosal immune response determined by secretory IgA, possibly related to active infection.

The development of synthetic peptides and the identification of H. pylori-specific epitopes, particularly CagA, and their use in immunodiagnosis, could be a significant contribution to the non-invasive prevention of gastric cancer, first-line diagnosis, and epidemiological studies, which are fundamental in high-risk populations with a high prevalence of infection.