Multi-Epitope Protein as a Tool of Serological Diagnostic Development for HTLV-1 and HTLV-2 Infections.

A multi-epitope protein expressed in a prokaryotic system, including epitopes of Env, Gag, and Tax proteins of both HTLV-1 and HTLV-2 was characterized for HTLV-1/2 serological screening. This tool can contribute to support the implementation of public policies to reduce HTLV-1/2 transmission in Brazil, the country with the highest absolute numbers of HTLV-1/2 infected individuals. The chimeric protein was tested in EIA using serum/plasma of HTLV-infected individuals and non-infected ones from four Brazilian states, including the North and Northeast regions (that present high prevalence of HTLV-1/2) and Southeast region (that presents intermediate prevalence rates) depicting different epidemiological context of HTLV-1/2 infection in our country. We enrolled samples from Pará (n = 114), Maranhão (n = 153), Minas Gerais (n = 225) and São Paulo (n = 59) states; they are from blood donors' candidates (Pará and Minas Gerais), pregnant women (Maranhão) and HIV+/high risk for sexually transmitted infection (STI; São Paulo). Among the HTLV-1/2 positive sera, there were co-infections with viral (HTLV-1 + HTLV-2, HIV, HCV, and HBV), bacterial (Treponema pallidum) and parasitic (Trypanosoma cruzi, Schistosma mansoni, Strongyloides stercoralis, Entamoeba coli, E. histolytica, and Endolimax nana) pathogens related to HTLV-1/2 co-morbidities that can contribute to inconclusive diagnostic results. Sera positive for HIV were included among the HTLV-1/2 negative samples. Considering both HTLV-1 and HTLV-2-infected samples from all states and different groups (blood donor candidates, pregnant women, and individuals with high risk for STI), mono or co-infected and HTLV-/HIV+, the test specificity ranged from 90.09 to 95.19% and the sensitivity from 82.41 to 92.36% with high accuracy (ROC AUC = 0.9552). This multi-epitope protein showed great potential to be used in serological screening of HTLV-1 and HTLV-2 in different platforms, even taking into account the great regional variation and different profile of HTLV-1 and HTLV-2 mono or co-infected individuals.

Multi-Epitope Protein as a Tool of Serological Diagnostic Development for HTLV-1 and HTLV-2 Infections

A multi-epitope protein expressed in a prokaryotic system, including epitopes of Env, Gag, and Tax proteins of both HTLV-1 and HTLV-2 was characterized for HTLV-1/2 serological screening. This tool can contribute to support the implementation of public policies to reduce HTLV-1/2 transmission in Brazil, the country with the highest absolute numbers of HTLV-1/2 infected individuals. The chimeric protein was tested in EIA using serum/plasma of HTLV-infected individuals and non-infected ones from four Brazilian states, including the North and Northeast regions (that present high prevalence of HTLV-1/2) and Southeast region (that presents intermediate prevalence rates) depicting different epidemiological context of HTLV-1/2 infection in our country. We enrolled samples from Pará (n = 114), Maranhão (n = 153), Minas Gerais (n = 225) and São Paulo (n = 59) states; they are from blood donors' candidates (Pará and Minas Gerais), pregnant women (Maranhão) and HIV+/high risk for sexually transmitted infection (STI; São Paulo). Among the HTLV-1/2 positive sera, there were co-infections with viral (HTLV-1 + HTLV-2, HIV, HCV, and HBV), bacterial (Treponema pallidum) and parasitic (Trypanosoma cruzi, Schistosma mansoni, Strongyloides stercoralis, Entamoeba coli, E. histolytica, and Endolimax nana) pathogens related to HTLV-1/2 co-morbidities that can contribute to inconclusive diagnostic results. Sera positive for HIV were included among the HTLV-1/2 negative samples. Considering both HTLV-1 and HTLV-2-infected samples from all states and different groups (blood donor candidates, pregnant women, and individuals with high risk for STI), mono or co-infected and HTLV-/HIV+, the test specificity ranged from 90.09 to 95.19% and the sensitivity from 82.41 to 92.36% with high accuracy (ROC AUC = 0.9552). Thismulti-epitope protein showed great potential to be used in serological screening of HTLV-1 and HTLV-2 in different platforms, even taking into account the great regional variation and different profile of HTLV-1 and HTLV-2 mono or co-infected individuals. (AU)

Cloning, expression, purification and functional analysis of a specific multi-epitope protein from multi drug resistance Acinetobacter baumannii.

BACKGROUND AND OBJECTIVES: Immunization is a promising strategy to combat against the life-threatening infections by Multi Drug Resistance Acinetobacter baumannii. In this study, we directed to design and evaluate the efficacy of a recombinant multi-epitope protein against this pathogen. MATERIALS AND METHODS: Epitopes prediction was performed for candidate proteins OmpA and BAM complex (BamA, BamB, BamC, BamD, BamE) from A. baumannii, using immune-informatics tools with high affinity for the human HLA alleles. After expression and purification of the recombinant protein, its functional activity was confirmed by interaction with positive sera. RESULTS: Cloning and expression of the desired multi-epitopes protein were verified. Circular Dichroism study showed the secondary structure and proper refolding of the recombinant protein was achieved and matched with computational prediction. There was a significant interaction between designed protein with antibodies presented in ICU patients' and staff's sera. CONCLUSION: The interaction of the recombinant protein with patients' sera antibodies suggests that it may be a promising determinant protein for immunization against of MDR A. baumannii.

Reverse and structural vaccinology approach to design a highly immunogenic multi-epitope subunit vaccine against Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a pathogen that resides in the upper respiratory tract of healthy individuals, maintaining a commensal relationship with its host. However, the virulent form may be the etiology of pneumonia, meningitis, bacteremia, and other respiratory tract infections. Streptococcal diseases are preventable by vaccination; but currently available vaccines have some drawbacks, especially due to the high capsule variability of streptococci strains. Thus, an effective prevention strategy continues to be the focus of extensive research. In our work, several bioinformatics tools were used to identify immunogenic peptides from a selected pool of 46 conserved proteins from Streptococcus pneumoniae. In silico analysis showed that 10 proteins had epitopes with affinity for B and T lymphocytes, which were present in at least 26 different pathogens serotypes and were considered promiscuous. The multi-epitope protein, designated HC44, was designed based on these epitopes and specific linkers to improve stability and exposure to T lymphocytes. The recombinant HC44 protein was expressed in E.coli and Swiss-Webster mice were immunised by intraperitoneal injection. Immunisation with the multi-epitope HC44 protein resulted in the production of very high levels of IgG with title superior to 1/1.200.000. However, subtype IgG was highly unbalanced toward IgG1 and no protection was afforded after challenge with S.pneumoniae in a sepsis model. Thus, our strategy has been effective in constructing a highly antigenic protein but novel immunisation strategies should be investigated to reorient the immune system toward a protective response.

Indirect ELISA using a multi-epitope recombinant protein to detect antibodies against foot-and-mouth disease virus serotype O in pigs.

Foot-and-mouth disease (FMD) is a devastating animal disease. A previously developed multi-epitope protein B4 vaccine of the FMD virus (FMDV) serotype O provides safety advantages over inactivated vaccines and could be used to prevent and control FMD in pigs. Commercial enzyme-linked immunosorbent assay (ELISA) kits for assessing vaccine efficacy are available for the inactivated vaccines but not for the multi-epitope protein vaccine. In this study, multi-epitope protein B4 was expressed in Escherichia coli, and an indirect ELISA (I-ELISA) was developed to detect antibodies against FMDV serotype O in pigs. The specificity and sensitivity were 96.7% and 95.9%, respectively. B4-vaccinated pigs yielded B4 I-ELISA serum values that were positively correlated with clinical protection against challenge with FMDV serotype O. The I-ELISA's ability to detect antibodies from animals vaccinated with the inactivated vaccine was also evaluated, and the B4 I-ELISA values were significantly positively correlated with liquid-phase blocking ELISA (LPBE) titers (r = 0.6708, p < 0.0001); thus, the I-ELISA was also suitable for detection of antibodies from swine vaccinated with the inactivated vaccine.

A recombinant multi-epitope protein MEP1 elicits efficient long-term immune responses against HIV-1 infection.

The effective protective HIV vaccine should elicit either protective antibodies or effective T cell response, or both. To improve the efficacy of HIV-1 vaccines, HLA polymorphism and HIV-1 diversity are 2 key factors to be considered for vaccine development. In this study, we expressed a recombinant multi-epitope protein MEP1 which has the same amino acid sequence as a DNA vaccine for Chinese population in our previous report. We found that MEP1 alone could elicit moderate levels of humoral and cellular immune responses, but these responses could not provide protection from challenge with a recombinant virus rTTV-lucgag, which expresses Gag of HIV-1 CRF_07BC. Nevertheless, when MEP1 was immunized with aluminum adjuvant, both humoral and cellular immune responses were significantly increased, and they were protective against virus infection; meanwhile, MEP1 with aluminum not only elicited early (10 d post immunization) but also a long-term (at least 44 weeks post immunization) immune responses in BALB/c mice. These results suggested that MEP1 has the potential to be developed as an effective vaccine candidate, and that suitable adjuvant is necessary for this protein to generate protective immune responses.

Rational design and efficacy of a multi-epitope recombinant protein vaccine against foot-and-mouth disease virus serotype A in pigs.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals, and outbreaks of this disease are often economically catastrophic. Recently, a series of outbreaks of foot-and-mouth disease virus (FMDV) serotype A occurred in many countries, including China. Therefore, it is necessary to develop safe and effective vaccines. We designed multi-epitope recombinant proteins A6, A7, and A8 with different three-dimensional structures and compared their immunogenicity in pigs. The results indicated that A8 conferred the greatest protection against FMDV serotype A challenge in pigs, and A8 was selected as the vaccine antigen. We further tested the adjuvant activity of CpG DNA in conjunction with the A8 vaccine, and the results showed significantly increased antigen-specific IFN-γ responses in pigs co-administered A8 with CpG compared to those vaccinated with A8 alone. A vaccine potency test showed that the CpG-adjuvanted A8 vaccine contained a 10.81 protective dose 50% (PD50) per dose for pigs, suggesting the potential for this vaccine to be used in emergency vaccination campaigns for the prevention of FMDV serotype A infection in pigs.

Evaluation of cross-protection against three topotypes of serotype O foot-and-mouth disease virus in pigs vaccinated with multi-epitope protein vaccine incorporated with poly(I:C).

Epitope-based vaccines are always questioned for their cross-protection against the antigenically variable foot-and-mouth disease virus (FMDV). In this study, we proved the cross-protection effect of a multi-epitope vaccine incorporated with poly(I:C) against three topotypes of O type FMDV. A total of 45 naïve pigs were vaccinated with different doses of multi-epitope protein vaccine incorporated with poly(I:C). At 28 days post-vaccination, 45 vaccinated and 6 unvaccinated control pigs (two pigs for each group) were challenged with three topotypes of virulent O type FMDV, namely, O/Mya/98 (Southeast Asia topotype), O/HN/CHA/93 (Cathay topotype) and O/Tibet/CHA/99 (PanAsia topotype) strains. All unvaccinated pigs developed generalised FMD clinical signs. Results showed that all pigs (n=15) conferred complete protection against the O/Mya/98 and O/HN/CHA/93 FMDV strains, 11 of which were protected against the O/Tibet/CHA/99 FMDV strain. The 50% protective dose values of the vaccine against the O/Mya/98, O/HN/CHA/93 and O/Tibet/CHA/99 FMDV strains were 15.59, 15.59 and 7.05, respectively. Contact challenge experiment showed that transmission occurred from the donors to the unvaccinated but not to vaccinated pigs. These results showed that vaccination with multi-epitope protein vaccine incorporated with poly(I:C) can efficiently prevent FMD in pigs.