In Silico and in Vivo Analysis of HIV-1 Rev Regulatory Protein for Evaluation of a Multiepitope-based Vaccine Candidate.

In silico-designed multiepitope conserved regions of human immunodeficiency virus 1 (HIV-1) proteins would be a beneficial strategy for antigen design which induces effective anti-HIV-1 T-cell responses. The conserved multiple HLA-DR-binding epitopes of Rev protein were identified using IEDB MHC-I prediction tools and SYFPEITHI webserver to screen potential T-cell epitopes. We analyzed toxicity, allergenicity, immunogenicity, hemolytic activity, cross-reactivity, cell-penetrating peptide (CPP) potency, and molecular docking of the candidate epitopes using several immune-informatics tools. Afterward, we designed a novel multiepitope construct based on non-toxic and non-allergenic Rev, Nef, Gp160 and P24-derived cytotoxic T cell (CTL) and T-helper cell (HTL) epitopes. Next, the designed construct (Nef-Rev-Gp160-P24) was subjected to three B-cell epitope prediction webservers, ProtParam and Protein-Sol to obtain the physicochemical features. Then, the recombinant multiepitope DNA and polypeptide constructs were complexed with different CPPs for nanoparticle formation and pass them via the cell membranes. Finally, the immunogenicity of multiepitope constructs in a variety of modalities was evaluated in mice. The results demonstrated that groups immunized with heterologous DNA+ MPG or HR9 CPP prime/rNef-Rev-Gp160-P24 polypeptide + LDP-NLS CPP boost regimens could significantly produce higher levels of IFN-γ and Granzyme B, and lower amounts of IL-10 than other groups. Moreover, higher levels of IgG2a and IgG2b were observed in all heterologous prime-boost regimens than homologous DNA or polypeptide regimens. Altogether, the present findings indicated that the Nef-Rev-Gp160-P24 polypeptide meets the criteria to be potentially useful as a multiepitope-based vaccine candidate against HIV-1 infection.

Immunological evaluation of two novel engineered Plasmodium vivax circumsporozoite proteins formulated with different human-compatible vaccine adjuvants in C57BL/6 mice.

A vaccine targeting Plasmodium vivax signifies an additional necessary tool when considering the malaria elimination/eradication goal. In this study, in vivo immunological evaluation of two novel engineered proteins of P. vivax circumsporozoite (PvCS127 and PvCS712) with two different arrangements of the repeat sequences of VK210 and VK247 was assessed. The immunological properties of the Escherichia coli-expressed chimeric proteins were evaluated by the immunization of C57BL/6 mice administered in NLX, CpG-ODNs, and QS21, alone or in combination as adjuvants. A significant increase in anti-rPvCS127 and -rPvCS712 IgG antibodies was observed in all the vaccine groups after the first boost, and the predominant isotypes were high-avidity cytophilic antibodies, IgG2b, and IgG2c. The highest ratio of IgG2b/IgG1 (2.74) and IgG2c/IgG1 (2.1) levels was detected in mouse groups immunized with rPvCS712 + NLX-CpG-QS21. The lowest level of IFN-γ (mean: 441 and 588 pg/mL, respectively) was produced by the mouse group, which received both antigens without any adjuvant, while significant levels of IFN-γ were detected in the mouse groups immunized with rPvCS127- or rPvCS712-NLX-CpG-QS21 formulation (mean: 1200 and 3092 pg/mL, respectively). The current results indicated that in C57BL/6 mice, both recombinant antigens were efficient immunogens and could induce humoral and cellular immune responses and their combination with three Th1 potent adjuvants had an impact on the magnitude and the quality of humoral responses (specific antibody subclasses, titer, and high avidity). Although the overall response was marginally higher for rPvCS712 than rPvCS127, all immunized mice induced some immune responses against both proteins, and the present findings indicate that rPvCS127 and rPvCS712 meet the criteria to be potentially useful vaccine candidates against P. vivax malaria.

Biological, immunological and functional properties of two novel multi-variant chimeric recombinant proteins of CSP antigens for vaccine development against Plasmodium vivax infection.

The circumsporozoite protein (CSP) of the malaria parasite Plasmodium vivax is a major pre-erythrocyte vaccine candidate. The protein has a central repeat region that belongs to one of repeat families (VK210, VK247, and the P. vivax-like). In the present study, computer modelling was employed to select chimeric proteins, comprising the conserved regions and different arrangements of the repeat elements (VK210 and VK247), whose structure is similar to that of the native counterparts. DNA encoding the selected chimeras (named CS127 and CS712) were synthetically constructed based on E. coli codons, then cloned and expressed. Mouse monoclonal antibodies (mAbs; anti-Pv-210-CDC and -Pv-247-CDC), recognized the chimeric antigens in ELISA, indicating correct conformation and accessibility of the B-cell epitopes. ELISA using IgG from plasma samples collected from 221 Iranian patients with acute P. vivax showed that only 49.32% of the samples reacted to both CS127 and CS712 proteins. The dominant subclass for the two chimeras was IgG1 (48% of the positive responders, OD492=0.777±0.420 for CS127; 48.41% of the positive responders, OD492=0.862±0.423 for CS712, with no statistically significant difference P>0.05; Wilcoxon signed ranks test). Binding assays showed that both chimeric proteins bound to immobilized heparan sulphate and HepG2 hepatocyte cells in a concentration-dependent manner, saturable at 80µg/mL. Additionally, anti-CS127 and -CS712 antibodies raised in mice recognized the native protein on the surface of P. vivax sporozoite with high intensity, confirming the presence of common epitopes between the recombinant forms and the native proteins. In summary, despite structural differences at the molecular level, the expression levels of both chimeras were satisfactory, and their conformational structure retained biological function, thus supporting their potential for use in the development of vivax-based vaccine.