Oncolytic herpes simplex viruses for the treatment of glioma and targeting glioblastoma stem-like cells.

Glioblastoma (GBM) is one of the most lethal cancers, having a poor prognosis and a median survival of only about 15 months with standard treatment (surgery, radiation, and chemotherapy), which has not been significantly extended in decades. GBM demonstrates remarkable cellular heterogeneity, with glioblastoma stem-like cells (GSCs) at the apex. GSCs are a subpopulation of GBM cells that possess the ability to self-renew, differentiate, initiate tumor formation, and manipulate the tumor microenvironment (TME). GSCs are no longer considered a static population of cells with specific markers but are quite flexible phenotypically and in driving tumor heterogeneity and therapeutic resistance. In light of these features, they are a critical target for successful GBM therapy. Oncolytic viruses, in particular oncolytic herpes simplex viruses (oHSVs), have many attributes for therapy and are promising agents to target GSCs. oHSVs are genetically-engineered to selectively replicate in and kill cancer cells, including GSCs, but not normal cells. Moreover, oHSV can induce anti-tumor immune responses and synergize with other therapies, such as chemotherapy, DNA repair inhibitors, and immune checkpoint inhibitors, to potentiate treatment effects and reduce GSC populations that are partly responsible for chemo- and radio-resistance. Herein, we present an overview of GSCs, activity of different oHSVs, clinical trial results, and combination strategies to enhance efficacy, including therapeutic arming of oHSV. Throughout, the therapeutic focus will be on GSCs and studies specifically targeting these cells. Recent clinical trials and approval of oHSV G47Δ in Japan for patients with recurrent glioma demonstrate the efficacy and promise of oHSV therapy.

Gene delivery in adherent and suspension cells using the combined physical methods.

Physical methods are widely utilized to deliver nucleic acids into cells such as electro-transfection or heat shock. An efficient gene electro-transfection requires the best conditions including voltage, the pulse length or number, buffer, incubation time and DNA form. In this study, the delivery of pEGFP-N1 vector into two adherent cell lines (HEK-293 T and COS-7) with the same origin (epithelial cells), and also mouse bone marrow-derived dendritic cells (DCs) was evaluated using electroporation under different conditions alone and along with heat treatment. Our data showed that the highest green fluorescent protein (GFP) expression in HEK-293 T and COS-7 cells was observed in serum-free RPMI cell culture medium as electroporation buffer, voltage (200 V), the pulse number (2), the pulse length (15 ms), the circular form of DNA, and 48 h after electro-transfection. In addition, the highest GFP expression in DCs was detected in serum-free RPMI, voltage (300 V), the pulse number (1), the pulse length (5 ms), and 48 h after electro-transfection. The use of sucrose as electroporation buffer, the pulse number (2), and the pulse length (25 ms) led to further cytotoxicity and lower transfection in HEK293T and COS-7 cells than other conditions. Moreover, the high voltage (700 V) increased the cell cytotoxicity, and decreased electro-transfection efficiency in DCs. On the other hand, the best conditions of electroporation along with heat treatment could significantly augment the transfection efficiency in all the cells. These data will be useful for gene delivery in other cells with the same properties using physical methods. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00524-4.

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.

The next generation of HCV vaccines: a focus on novel adjuvant development.

INTRODUCTION: Considerable efforts have been made to treat and prevent acute and chronic infections caused by the hepatitis C virus (HCV). Current treatments are unable to protect people from reinfection. Hence, there is a need for development of both preventive and therapeutic HCV vaccines. Many vaccine candidates are in development to fight against HCV, but their efficacy has so far proven limited partly due to low immunogenicity. AREAS COVERED: We explore development of novel and powerful adjuvants to achieve an effective HCV vaccine. The basis for developing strong adjuvants is to understand the innate immunity pathway, which subsequently stimulates humoral and cellular immune responses. We have also investigated immunogenicity of developed adjuvants that have been used in recent studies available in online databases such as PubMed, PMC, ScienceDirect, Google Scholar, etc. EXPERT OPINION: Adjuvants are used as a part of vaccine formulation to boost vaccine immunogenicity and antigen delivery. Several FDA-approved adjuvants are used in licensed human vaccines. Unfortunately, no adjuvant has yet been proven to boost HCV immune responses to the extent needed for an effective vaccine. One of the promising approaches for developing an effective adjuvant is the combination of various adjuvants to trigger several innate immune responses, leading to activation of adaptive immunity.[Figure: see text].

In silico design and in vitro expression of novel multiepitope DNA constructs based on HIV-1 proteins and Hsp70 T-cell epitopes.

OBJECTIVES: Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection. METHODS: Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting. RESULTS: The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56-60% as the bands of ~ 63 and ~ 72 kDa confirmed in western blotting, respectively. CONCLUSION: The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development.

Antimicrobial/anticancer peptides: bioactive molecules and therapeutic agents.

Antimicrobial peptides (AMPs) have been known as host-defense peptides. These cationic and amphipathic peptides are relatively short (∼5-50 L-amino acids) with molecular weight less than 10 kDa. AMPs have various roles including immunomodulatory, angiogenic and antitumor activities. Anticancer peptides (ACPs) are a main subset of AMPs as a novel therapeutic approach against tumor cells. The physicochemical properties of the ACPs influence their cell penetration, stability and efficiency of targeting. Up to now, several databases and web servers for in silico prediction of AMPs/ACPs have been established prior to the lab analysis. The present review focuses on the recent advancement about AMPs/ACPs activities including their in silico prediction by computational tools and their potential applications as therapeutic agents especially in cancer.

Exploring novel and potent cell penetrating peptides in the proteome of SARS-COV-2 using bioinformatics approaches.

Among various delivery systems for vaccine and drug delivery, cell-penetrating peptides (CPPs) have been known as a potent delivery system because of their capability to penetrate cell membranes and deliver some types of cargoes into cells. Several CPPs were found in the proteome of viruses such as Tat originated from human immunodeficiency virus-1 (HIV-1), and VP22 derived from herpes simplex virus-1 (HSV-1). In the current study, a wide-range of CPPs was identified in the proteome of SARS-CoV-2, a new member of coronaviruses family, using in silico analyses. These CPPs may play a main role for high penetration of virus into cells and infection of host. At first, we submitted the proteome of SARS-CoV-2 to CellPPD web server that resulted in a huge number of CPPs with ten residues in length. Afterward, we submitted the predicted CPPs to C2Pred web server for evaluation of the probability of each peptide. Then, the uptake efficiency of each peptide was investigated using CPPred-RF and MLCPP web servers. Next, the physicochemical properties of the predicted CPPs including net charge, theoretical isoelectric point (pI), amphipathicity, molecular weight, and water solubility were calculated using protparam and pepcalc tools. In addition, the probability of membrane binding potential and cellular localization of each CPP were estimated by Boman index using APD3 web server, D factor, and TMHMM web server. On the other hand, the immunogenicity, toxicity, allergenicity, hemolytic potency, and half-life of CPPs were predicted using various web servers. Finally, the tertiary structure and the helical wheel projection of some CPPs were predicted by PEP-FOLD3 and Heliquest web servers, respectively. These CPPs were divided into: a) CPP containing tumor homing motif (RGD) and/or tumor penetrating motif (RXXR); b) CPP with the highest Boman index; c) CPP with high half-life (~100 hour) in mammalian cells, and d) CPP with +5.00 net charge. Based on the results, we found a large number of novel CPPs with various features. Some of these CPPs possess tumor-specific motifs which can be evaluated in cancer therapy. Furthermore, the novel and potent CPPs derived from SARS-CoV-2 may be used alone or conjugated to some sequences such as nuclear localization sequence (NLS) for vaccine and drug delivery.

Cppsite 2.0: An Available Database of Experimentally Validated Cell-Penetrating Peptides Predicting their Secondary and Tertiary Structures.

One of the biggest barriers in drug and vaccine development is to find an effective delivery system. Cell-penetrating peptides (CPPs) play a crucial role for delivery of biological cargoes and pass them through the membranes. Several databases have been developed for therapeutic peptides as potential drug candidates and delivery vehicles. A rapid growth has occurred in many patents and research articles on CPPs as therapeutic peptides. To save time and cost in laboratories, prediction and design of CPPs before in vitro/in vivo experiments using computational methods and online web servers are rational. Various online web servers which provide prediction of CPPs including CellPPD, CPPpred, CPPred-RF and MLCPP, and also different curated databases that present validated information of CPPs such as CPPsite 2.0 have been developed up to now. Two methods including CellPPD and CPPpred were applied to predict and design potent CPPs. CPPsite 2.0 is a user-friendly updated database that provides various information about CPPs and contains 1855 entries. This database provides comprehensive information on experimentally tested CPPs and prediction of their secondary and tertiary structures to realize their structure-function relationship. Furthermore, each entry presents information of a CPP including chirality, origin, nature of peptide, sub-cellular localization, uptake mechanism and efficiency, amino acid composition, hydrophobicity, and physicochemical properties. One of main goals of CPPsite 2.0 database is to provide the latest datasets of CPPs for analysis and development of CPP prediction methods. CPPsite 2.0 is freely available at https://webs.iiitd.edu.in/raghava/cppsite.

Antiviral therapy for the sexually transmitted viruses: recent updates on vaccine development.

INTRODUCTION: The sexually transmitted infections (STIs) caused by viruses including human T cell leukemia virus type-1 (HTLV-1), human immunodeficiency virus-1 (HIV-1), human simplex virus-2 (HSV-2), hepatitis C virus (HCV), hepatitis B virus (HBV), and human papillomavirus (HPV) are major public health issues. These infections can cause cancer or result in long-term health problems. Due to high prevalence of STIs, a safe and effective vaccine is required to overcome these fatal viruses. AREAS COVERED: This review includes a comprehensive overview of the literatures relevant to vaccine development against the sexually transmitted viruses (STVs) using PubMed and Sciencedirect electronic search engines. Herein, we discuss the efforts directed toward development of effective vaccines using different laboratory animal models including mice, guinea pig or non-human primates in preclinical trials, and human in clinical trials with different phases. EXPERT OPINION: There is no effective FDA approved vaccine against the sexually transmitted viruses (STVs) except for HBV and HPV as prophylactic vaccines. Many attempts are underway to develop vaccines against these viruses. There are several approaches for improving prophylactic or therapeutic vaccines such as heterologous prime/boost immunization, delivery system, administration route, adjuvants, etc. In this line, further studies can be helpful for understanding the immunobiology of STVs in human. Moreover, development of more relevant animal models is a worthy goal to induce effective immune responses in humans.

An overview of in silico vaccine design against different pathogens and cancer.

INTRODUCTION: Due to overcome the hardness of the vaccine design, computational vaccinology is emerging widely. Prediction of T cell and B cell epitopes, antigen processing analysis, antigenicity analysis, population coverage, conservancy analysis, allergenicity assessment, toxicity prediction, and protein-peptide docking are important steps in the process of designing and developing potent vaccines against various viruses and cancers. In order to perform all of the analyses, several bioinformatics tools and online web servers have been developed. Scientists must take the decision to apply more suitable and precise servers for each part based on their accuracy. AREAS COVERED: In this review, a wide-range list of different bioinformatics tools and online web servers has been provided. Moreover, some studies were proposed to show the importance of various bioinformatics tools for predicting and developing efficient vaccines against different pathogens including viruses, bacteria, parasites, and fungi as well as cancer. EXPERT OPINION: Immunoinformatics is the best way to find potential vaccine candidates against different pathogens. Thus, the selection of the most accurate tools is necessary to predict and develop potent preventive and therapeutic vaccines. To further evaluation of the computational and in silico vaccine design, in vitro/in vivo analyses are required to develop vaccine candidates.

B1 protein: a novel cell penetrating protein for in vitro and in vivo delivery of HIV-1 multi-epitope DNA constructs.

OBJECTIVES: Enhancement of the potential ability of biomacromolecules to cross cell membranes is a critical step for development of effective therapeutic vaccine especially DNA vaccine against human immunodeficiency virus-1 (HIV-1) infection. The supercharged proteins were known as powerful weapons for delivery of different types of cargoes such as DNA and protein. Hence, we applied B1 protein with + 43 net charges obtained from a single frameshift in the gene encoding enhanced green fluorescent protein (eGFP) for delivery of two multi-epitope DNA constructs (nef-vpu-gp160-p24 and nef-vif-gp160-p24) in vitro and in vivo for the first time. For this purpose, B1 protein was generated in bacterial expression system under native conditions, and used to interact with both DNA constructs. RESULTS: Our data indicated that B1 protein (~ 27 kDa) was able to form a stable nanoparticle (~ 80-110 nm) with both DNA constructs at nitrogen: phosphate (N: P) ratio of 1:1. Moreover, the transfection efficiency of B1 protein for DNA delivery into HEK-293T cell line indicated that the cellular uptake of nef-vif-gp160-p24 DNA/ B1 and nef-vpu-gp160-p24 DNA/ B1 nanoparticles was about 32-35% with lower intensity as compared to TurboFect commercial reagent. On the other hand, immunization of BALB/c mice with different modalities demonstrated that B1 protein could enhance the levels of antibody, IFN-gamma and Granzyme B eliciting potent and strong Th1-directed cellular immunity. CONCLUSION: Generally, our findings showed the potency of B1 protein as a promising gene delivery system to improve an effective therapeutic vaccine against HIV-1 infection.

Comparative analysis of two HIV-1 multiepitope polypeptides for stimulation of immune responses in BALB/c mice.

A licensed vaccine against human immunodeficiency virus-1 (HIV-1) infection has not become available up to now. Hence, it is more rational to use immune-informatics tools for prediction of T cell epitopes (in silico study) and development of an effective epitope-driven vaccine against hypervariable pathogens. Multiepitope vaccines were considered as the next generation of an effective vaccine against HIV-1 infection. In the current study, we developed two different constructs encoding T cell epitopes derived from Nef, Vif, Vpu, Gp160 and P24 proteins in BALB/c mice. To overcome their poor immunogenicity, four different cell penetrating peptides (MPG and HR9 for DNA delivery, and CyLoP-1 and LDP-NLS for protein delivery), Montanide adjuvant, and heterologous prime-boost immunization strategy were utilized. The generation of cytokines, Granzyme B, and total IgG and its subclasses was determined using ELISA. Our data indicated that the levels of IFN-γ and Granzyme B in mice injected with Nef-Vif-Gp160-P24 multiepitope constructs were higher than those immunized with Nef-Vpu-Gp160-P24 multiepitope constructs. Moreover, the heterologous DNA priming/ multiepitope peptide boosting in both Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 regimens induced significantly high antigen-specific IgG2a and IgG2b responses in comparison with other groups. There was no significant difference between MPG and HR9 as well as CyLoP-1 and LDP-NLS as a delivery system for enhancement of immune responses. Generally, the heterologous DNA prime/ multiepitope peptide boost modalities for both constructs could significantly enhance the levels of IgG2a, IgG2b, IFN-γ, and Granzyme B directed toward Th1 immune responses as compared to homologous prime/ boost with DNA or polypeptide constructs.

Comparison of HIV-1 Vif and Vpu accessory proteins for delivery of polyepitope constructs harboring Nef, Gp160 and P24 using various cell penetrating peptides.

To develop an effective therapeutic vaccine against HIV-1, prediction of the most conserved epitopes derived from major proteins using bioinformatics tools is an alternative achievement. The epitope-driven vaccines against variable pathogens represented successful results. Hence, to overcome this hyper-variable virus, we designed the highly conserved and immunodominant peptide epitopes. Two servers were used to predict peptide-MHC-I binding affinity including NetMHCpan4.0 and Syfpeithi servers. The NetMHCIIpan3.2 server was utilized for MHC-II binding affinity. Then, we determined immunogenicity scores and allergenicity by the IEDB immunogenicity predictor and Algpred, respectively. Next, for estimation of toxicity and population coverage, ToxinPred server and IEDB population coverage tool were applied. After that, the MHC-peptide binding was investigated by GalexyPepDock peptide-protein flexible docking server. Finally, two different DNA and peptide constructs containing Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 were prepared and complexed with four various cell penetrating peptides (CPPs) for delivery into mammalian cells (MPG and HR9 CPPs for DNA delivery, and CyLoP-1 and LDP-NLS CPPs for protein delivery). Our results indicated that the designed DNA and peptide constructs could form non-covalent stable nanoparticles at certain ratios as observed by scanning electron microscope (SEM) and Zetasizer. The flow cytometry results obtained from in vitro transfection of the nanoparticles into HEK-293T cell lines showed that the percentage of GFP expressing cells was about 38.38 ± 1.34%, 25.36% ± 0.30, 54.95% ± 0.84, and 25.11% ± 0.36 for MPG/pEGFP-nef-vif-gp160-p24, MPG/pEGFP-nef-vpu-gp160-p24, HR9/pEGFP-nef-vif-gp160-p24 and HR9/pEGFP-nef-vpu-gp160-p24, respectively. Thus, these data showed that the DNA construct harboring nef-vif-gp160-p24 multi-epitope gene had higher efficiency than the DNA construct harboring nef-vpu-gp160-p24 multi-epitope gene to penetrate into the cells. Moreover, delivery of the recombinant Nef-Vif-Gp160-P24 and Nef-Vpu-Gp160-P24 polyepitope peptides in HEK-293T cells was confirmed as a single band about 32 kDa using western blot analysis. Although, both DNA and peptide constructs could be successfully transported by a variety of CPPs into the cells, but the difference between them in transfection rate will influence the levels of immune responses for development of therapeutic vaccines.

Cell penetrating peptides: the potent multi-cargo intracellular carriers.

Introduction: Cell penetrating peptides (CPPs) known as protein translocation domains (PTD), membrane translocating sequences (MTS), or Trojan peptides (TP) are able to cross biological membranes without clear toxicity using different mechanisms, and facilitate the intracellular delivery of a variety of bioactive cargos. CPPs could overcome some limitations of drug delivery and combat resistant strains against a broad range of diseases. Despite delivery of different therapeutic molecules by CPPs, they lack cell specificity and have a short duration of action. These limitations led to design of combined cargo delivery systems and subsequently improvement of their clinical applications. Areas covered: This review covers all our studies and other researchers in different aspects of CPPs such as classification, uptake mechanisms, and biomedical applications. Expert opinion: Due to low cytotoxicity of CPPs as compared to other carriers and final degradation to amino acids, they are suitable for preclinical and clinical studies. Generally, the efficiency of CPPs was suitable to penetrate the cell membrane and deliver different cargos to specific intracellular sites. However, no CPP-based therapeutic approach has approved by FDA, yet; because there are some disadvantages for CPPs including short half-life in blood, and nonspecific CPP-mediated delivery to normal tissue. Thus, some methods were used to develop the functions of CPPs in vitro and in vivo including the augmentation of cell specificity by activatable CPPs, specific transport into cell organelles by insertion of corresponding localization sequences, incorporation of CPPs into multifunctional dendrimeric or liposomal nanocarriers to improve selectivity and efficiency especially in tumor cells.

Prediction of cross-clade HIV-1 T-cell epitopes using immunoinformatics analysis.

Epitope mapping has emerged as a powerful tool to develop peptide vaccines against hypervariable viruses such as HIV. This method has led to stimulate a specific immune response and achieve advanced vaccine formulations. In this study, we identified peptides that were potentially immunostimulatory and highly conserved in HIV-1 main group. The analyses included were CTL assay, Tap transport, and the potential allergenicity. The highest population coverage rate was also found for all potential T-cell epitopes in 16 specified geographic regions of the world. The current study is the first attempt to explore peptide-protein flexible docking across all the major epitopes of HIV-1. Our data indicated that REV54-63 and VPU58-66 with the highest epitope identification scores, GP16037-46 and VPR38-47 with the highest conservation (98.89%), and NEF134-144 and GP16037-46 epitopes with a higher quality of peptide-protein interaction models in docking procedure were chosen as putative epitopes among all HLA class I epitopes. TAT40-67 , VPR65-82 , and VPU30-44 with the highest score of binding affinity, VPR65-82 with the highest conservation (97.55%), and GP160481-498 epitope with a higher quality of peptide-protein interaction models in docking procedure were determined as putative epitopes among all HLA-DR epitopes. Furthermore, two epitopes of GP160481-498 and VIF144-159 were predicted to bind 22 and 21 HLA-II alleles, respectively. Accumulative population coverage of potential helper T-cell epitopes and CTL epitopes varied between 90.82% and 100%. Generally, these predicted highly immunogenic T-cell epitopes can contribute to design HIV-1 peptide vaccine candidates. Combination of bioinformatics tools with in vivo methods will be necessary for HIV-1 vaccine development.

Prime-boost vaccine strategy against viral infections: Mechanisms and benefits.

The essential goal of vaccination is to generate potent and long-term protection against diseases. Among different vaccine modalities, prime-boost vaccine strategies could enhance cellular and also humoral immunity in several animal models. These strategies have been applied for the development of vaccines against important infectious diseases such as HIV, SIV, HCV, HSV, and HBV indicating promising results even in clinical trials. Several factors including selection of antigen, type of vector, delivery route, dose, adjuvant, boosting regimen, the order of vector injection, and the intervals between different vaccinations influence the outcome of prime-boost immunization approaches. The reported data suggest that the prime-boost strategy as a combination of vaccines (i.e., heterologous prime-boost) may be better than a single vaccine for protection against infectious diseases. Indeed, in many cases, heterologous prime-boost can be more immunogenic than homologous prime-boost strategy. This review discusses the recent advances in prime-boost immunization strategies as well as their benefits and mechanisms of action.

Prime/boost immunization with HIV-1 MPER-V3 fusion construct enhances humoral and cellular immune responses.

Development of an effective vaccine against HIV-1 infection is a main concern in worldwide. A potent vaccine for HIV-1 requires the induction and maintenance of both humoral and cellular immunity. In this study, the levels of humoral and cellular immune responses were compared using MPER-V3 injection in three immunization strategies such as DNA/DNA, peptide/peptide, and DNA/peptide (prime-boost). MPG peptide and Montanide 720 were used as a DNA delivery system, and as a peptide adjuvant, respectively. Our results demonstrated that MPG forms stable non-covalent nanoparticles with plasmid DNA at N/P ratio of 10:1 (∼ 110-130 nm). The in vitro transfection efficiency of MPER-V3 DNA using MPG was comparable with lipofectamine and turbofect reagents as a common delivery system. In vivo prime-boost immunization using HIV-1 MPER-V3 could significantly enhance humoral and cellular immune responses as compared to control groups. The mixture of IgG1 and IgG2a was observed for each strategy, but IFN-γ production was significantly higher in prime-boost and peptide immunizations than that in DNA immunizations, inducing Th1 response. Moreover, our data showed that prime immunization with low dose of the nanoparticles (MPER-V3 DNA: MPG at ratio of 1:10) followed by MPER-V3 peptide drives T cell responses towards a Th1-type similar to high dose of the naked DNA prime/peptide boost immunization. Generally, the prime-boost strategy could improve both immune responses against MPER and especially V3 peptides suggesting its application as a promising HIV vaccine candidate in future.