Advances in cell-based delivery of oncolytic viruses as therapy for lung cancer.

Lung cancer's intractability is enhanced by its frequent resistance to (chemo)therapy and often high relapse rates that make it the leading cause of cancer death worldwide. Improvement of therapy efficacy is a crucial issue that might lead to a significant advance in the treatment of lung cancer. Oncolytic viruses are desirable combination partners in the developing field of cancer immunotherapy due to their direct cytotoxic effects and ability to elicit an immune response. Systemic oncolytic virus administration through intravenous injection should ideally lead to the highest efficacy in oncolytic activity. However, this is often hampered by the prevalence of host-specific, anti-viral immune responses. One way to achieve more efficient systemic oncolytic virus delivery is through better protection against neutralization by several components of the host immune system. Carrier cells, which can even have innate tumor tropism, have shown their appropriateness as effective vehicles for systemic oncolytic virus infection through circumventing restrictive features of the immune system and can warrant oncolytic virus delivery to tumors. In this overview, we summarize promising results from studies in which carrier cells have shown their usefulness for improved systemic oncolytic virus delivery and better oncolytic virus therapy against lung cancer.

In silico designing of novel epitope-based peptide vaccines against HIV-1.

The HIV-1 virus has been regarded as a catastrophe for human well-being. The global incidence of HIV-1-infected individuals is increasing. Hence, development of effective immunostimulatory molecules has recently attracted an increasing attention in the field of vaccine design against HIV-1 infection. In this study, we explored the impacts of CD40L and IFN-γ as immunostimulatory adjuvants for our candidate HIV-1 Nef vaccine in human and mouse using immunoinformatics analyses. Overall, 18 IFN-γ-based vaccine constructs (9 constructs in human and 9 constructs in mouse), and 18 CD40L-based vaccine constructs (9 constructs in human and 9 constructs in mouse) were designed. To find immunogenic epitopes, important characteristics of each component (e.g., MHC-I and MHC-II binding, and peptide-MHC-I/MHC-II molecular docking) were determined. Then, the selected epitopes were applied to create multiepitope constructs. Finally, the physicochemical properties, linear and discontinuous B cell epitopes, and molecular interaction between the 3D structure of each construct and CD40, IFN-γ receptor or toll-like receptors (TLRs) were predicted. Our data showed that the full-length CD40L and IFN-γ linked to the N-terminal region of Nef were capable of inducing more effective immune response than multiepitope vaccine constructs. Moreover, molecular docking of the non-allergenic full-length- and epitope-based CD40L and IFN-γ constructs to their cognate receptors, CD40 and IFN-γ receptors, and TLRs 4 and 5 in mouse were more potent than in human. Generally, these findings suggest that the full forms of these adjuvants could be more efficient for improvement of HIV-1 Nef vaccine candidate compared to the designed multiepitope-based constructs.

Development of novel HPV therapeutic vaccine constructs based on engineered exosomes and tumor cell lysates.

AIMS: Human papillomavirus (HPV) infections are highly prevalent globally. While preventive HPV vaccines exist, therapeutic vaccines are needed to treat existing HPV lesions and malignancies. This study evaluated the immunostimulatory and anti-tumor effects of three therapeutic vaccine candidates based on the recombinant protein, tumor cell lysate (TCL), and engineered exosome (Exo) harboring the heat shock protein 27 (Hsp27)-E7 fusion construct in mouse model. MAIN METHODS: At first, the recombinant Hsp27-E7 protein was generated in E. coli expression system. Then, tumor cell lysates-based and engineered exosomes-based vaccine constructs harboring green fluorescent protein (GFP) and Hsp27-E7 were produced using lentiviral system. Finally, their immunological and antitumor effects were investigated in both prophylactic and therapeutic experiments. KEY FINDINGS: Our data showed that the recombinant Hsp27-E7 protein, TCL-Hsp27-E7 and Exo-Hsp27-E7 regimens can induce the highest level of IFN-γ, TNF-α and Granzyme B, respectively. The percentage of tumor-free mice was identical for three vaccine strategies (survival rate: 75 %) in both prophylactic and therapeutic experiments. Generally, the TCL-Hsp27-E7, Exo-Hsp27-E7 and recombinant Hsp27-E7 protein regimens induced effective immune responses toward Th1 and CTL activity, and subsequently antitumor effects in mouse model. SIGNIFICANCE: Regarding to higher Granzyme B secretion, lower tumor growth and more safety, the Exo-Hsp27-E7 regimen can be considered as the most promising HPV vaccination strategy.

Immunostimulatory effects of Hsp70 fragments and Hsp27 in design of novel HIV-1 vaccine formulations.

BACKGROUND: Heat shock proteins (HSPs) as an adjuvant induce antigen-specific immunity through facilitating antigen presentation and stimulating T cells. In this study, the immunostimulatory properties of two major fragments of Hsp70 (N-Hsp70(aa 1-387) with ATPase property and C-Hsp70 (aa 508-641) with peptide-binding capacity) and the full length of Hsp27 as vaccine adjuvants were evaluated to boost HIV-1 Nef antigen-specific immunity in both in vitro and in vivo experiments. METHODS: At first, the nanoparticles harbouring DNA fusion constructs (i.e. N-Hsp70-Nef, C-Hsp70-Nef and Hsp27-Nef) complexed with HIV Rev (34-50) cell-penetrating peptide were generated to deliver DNA into the cells. Then, the recombinant Nef, Hsp27-Nef, N-Hsp70-Nef and C-Hsp70-Nef proteins were generated in E.coli expression system. Next, the immunostimulatory properties of these fusion constructs were evaluated in both in vitro and in vivo studies. Finally, the secretion of main cytokines from single-cycle replicable (SCR) HIV-1 virion-exposed splenocytes was investigated. RESULTS: Our data showed that the stable and non-toxic DNA/Rev nanoparticles could successfully deliver the genes of interest into the cells. Moreover, higher secretion of antibodies and cytokines was detected in mice receiving the Hsp-Nef constructs than in mice receiving Nef antigen. The C-Hsp70 was also superior for inducing Nef-specific Th1 and CTL immunity compared with N-Hsp70 and Hsp27. The T-cell activity was maintained in the SCR-exposed splenocytes, especially the splenocytes of mice receiving the C-Hsp70-Nef regimen. CONCLUSION: Altogether, these findings demonstrate the significance of Hsps as enhancers of antigen-specific immunity. Notably, the C-Hsp70 region showed better adjuvant properties for inducing cellular immunity in the improvement of HIV-1 therapeutic vaccines.

Investigation of Immunostimulatory Effects of IFN-γ Cytokine and CD40 Ligand Costimulatory Molecule for Development of HIV-1 Therapeutic Vaccine Candidate.

The most crucial disadvantage of DNA-based vaccines is their low immunogenicity; therefore, finding an effectual adjuvant is essential for their development. Herein, immunostimulatory effects of IFNγ cytokine and a CD40 ligand (CD40L) costimulatory molecule are evaluated as combined with an antigen, and also linked to an antigen in mice. For this purpose, after preparation of the HIV-1 Nef, IFNγ, and CD40L DNA constructs, and also their recombinant protein in an Escherichia coli expression system, nine groups of female BALB/c mice are immunized with different regimens of DNA constructs. About 3 weeks and also 3 months after the last injection, humoral and cellular immune responses are assessed in mice sera and splenocytes. Additionally, mice splenocytes are exposed to single-cycle replicable (SCR) HIV-1 virions for evaluating their potency in the secretion of cytokines in vitro. The data indicate that the linkage of IFNγ and CD40L to Nef antigen can significantly induce the Th-1 pathway and activate cytotoxic T lymphocytes compared to other regimens. Moreover, groups receiving the IFNγ-Nef and CD40L-Nef fusion DNA constructs show higher secretion of IFNγ and TNF-α from virion-infected lymphocytes than other groups. Therefore, the IFNγ-Nef and CD40L-Nef fusion DNA constructs are suggested to be a potential option for development of an efficient HIV-1 vaccine.

Engineered exosomes: a promising vehicle in cancer therapy.

During the past few decades, researchers have attempted to discover an effective treatment for cancer. Exosomes are natural nanovesicles released by various cells and play a role in communication between cells. While natural exosomes have high clinical potential, their inherent limitations have prompted researchers to design exosomes with improved therapeutic properties. To achieve this purpose, researchers have undertaken exosome engineering to modify the surface properties or internal composition of exosomes. After these modifications, engineered exosomes can be used as carriers for delivery of chemotherapeutic agents, targeted drug delivery or development of cancer vaccines. The present study provides an overview of exosomes, including their biogenesis, biological functions, isolation techniques, engineering methods, and potential applications in cancer therapy.

HPV co-infections with other pathogens in cancer development: A comprehensive review.

High-risk human papillomaviruses (HR-HPVs) cause various malignancies in the anogenital and oropharyngeal regions. About 70% of cervical and oropharyngeal cancers are caused by HPV types 16 and 18. Notably, some viruses including herpes simplex virus, Epstein-Barr virus, and human immunodeficiency virus along with various bacteria often interact with HPV, potentially impacting its replication, persistence, and cancer progression. Thus, HPV infection can be significantly influenced by co-infecting agents that influence infection dynamics and disease progression. Bacterial co-infections (e.g., Chlamydia trachomatis) along with bacterial vaginosis-related species also interact with HPV in genital tract leading to viral persistence and disease outcomes. Co-infections involving HPV and diverse infectious agents have significant implications for disease transmission and clinical progression. This review explores multiple facets of HPV infection encompassing the co-infection dynamics with other pathogens, interaction with the human microbiome, and its role in disease development.

Antiviral Peptides Derived from Plants: Their Designs and Functions.

In recent years, plant-derived bioactive compounds have been developed as antiviral agents. Plants synthesize a variety of compounds, especially peptides, which possess antimicrobial activity. Current studies have shown that some antimicrobial peptides have antiviral activity against a wide range of human DNA and RNA viruses and play an effective role in the treatment of human viral diseases. These peptides act through different mechanisms. They can integrate into the envelope of the target virus or cell membrane of the host, resulting in an unstable membrane. For instance, some peptides prevent the attachment of viral spike proteins to host cells. On the other hand, some peptides may alter the cellular pathways, including DNA replication or protein synthesis, leading to the suppression of viral infection. However, the antiviral activity of peptides can be affected by their chemical and structural properties. In several studies, the properties of antimicrobial (antiviral) peptides were altered by minor modifications, but these changes require tools to predict. Recently, computational approaches have been introduced to analyze the effects of structural modifications on the physicochemical properties, mechanism of action, stability, and activity of peptides. In this mini-review, we will describe the design and function of antiviral peptides derived from plants.

Recent advances in various bio-applications of bacteria-derived outer membrane vesicles.

Outer membrane vesicles (OMVs) are spherical nanoparticles released from gram-negative bacteria. OMVs were originally classified into native 'nOMVs' (produced naturally from budding of bacteria) and non-native (produced by mechanical means). nOMVs and detergent (dOMVs) are isolated from cell supernatant without any detergent cell disruption techniques and through detergent extraction, respectively. Growth stages and conditions e.g. different stress factors, including temperature, nutrition deficiency, and exposure to hazardous chemical agents can affect the yield of OMVs production and OMVs content. Because of the presence of bacterial antigens, pathogen-associated molecular patterns (PAMPs), various proteins and the vesicle structure, OMVs have been developed in many biomedical applications. OMVs due to their size can be phagocytized by APCs, enter lymph vessels, transport antigens efficiently, and induce both T and B cells immune responses. Non-engineered OMVs have been frequently used as vaccines against different bacterial and viral infections, and various cancers. OMVs can also be used in combination with different antigens as an attractive vaccine adjuvant. Indeed, foreign antigens from target microorganisms can be trapped in the lumen of nonpathogenic vesicles or can be displayed on the surface through bacterial membrane protein to increase the immunogenicity of the antigens. In this review, different factors affecting OMV production including time of cultivation, growth media, stress conditions and genetic manipulations to enhance vesiculation will be described. Furthermore, recent advances in various biological applications of OMVs such as vaccine, drug delivery, cancer therapy, and enzyme carrier are discussed. Generally, the application of OMVs as vaccine carrier in three categories (i.e., non-engineered OMVs, OMVs as an adjuvant, recombinant OMVs (rOMVs)), as delivery system for small interfering RNA and therapeutic agents, and as enzymes carrier will be discussed.

Immunoinformatics studies of heat shock proteins 27 and 70: Development of potent therapeutic vaccine constructs against human papillomavirus-related cancers.

Heat shock proteins (HSPs) improve cross-presentation of linked tumor antigens, thus they can be exploited in therapeutic vaccine design. Herein, in silico analyses of different vaccine constructs were performed based on human papillomavirus (HPV)-16 E7 protein linked to Homo sapiens/Mus musculus Hsp27 or Hsp70 in multiepitope and whole sequence forms. Then, computational comparison between different orientations of Hsp/E7 was carried out in both forms. Finally, molecular docking was performed between the designed constructs and signaling (TLRs) or endocytic (CD14, LOX-1 and SREC-1) receptors. Our data represented the high-ranked T-cell epitopes and the potential B-cell epitopes of Homo sapiens/Mus musculus Hsp27 and Hsp70. Moreover, molecular docking showed that whole sequence of Hsp27 had better interaction with all receptors than whole sequence of Hsp70 suggesting likely stronger stimulation of innate and adaptive immunity. All designed Homo sapiens/Mus musculus Hsp27/E7 constructs had better docking scores with the endocytic receptors especially SREC-1 than all designed Homo sapiens/Mus musculus Hsp70/E7 constructs in both orientations. Generally, the multiepitope-/whole sequence-based Homo sapiens/Mus musculus Hsp27-E7 fusion constructs showed more conservancy and immunogenicity than other designed constructs. These fusion constructs were non-allergenic, non-toxic and stable suggesting them as promising vaccine candidates against HPV-related cancers.

Systemic delivery of specific and efficient CRISPR/Cas9 system targeting HPV16 oncogenes using LL-37 antimicrobial peptide in C57BL/6 mice.

Human papillomavirus (HPV) type 16 is the most common sexually transmitted virus related to cervical cancer. Among different types of advanced novel therapies, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-mediated gene editing holds great promise for cancer treatment. In this research, optimal gRNA sequences targeting HPV16 E5, E6, E7, and p97 promoter for CRISPR/Cas9-mediated genome editing were designed by in silico prediction. After cloning, delivery of the recombinant vectors into C3, TC1 and HeLa tumor cells was evaluated by Lipofectamine 2000, and LL-37 antimicrobial peptide. Then, the levels of cell cycle proteins (p21, p53, and Rb) were investigated after treatment by western blot analysis. Finally, C57BL/6 mice were inoculated with C3 tumor cells, and treated with recombinant vectors and cisplatin. Based on the tumor size reduction and IHC results, the E6 + E7-treated group with a high percentage of cleaved caspase-3 positive cells (45.75%) and low mitotic index of 2-3 was determined as the best treatment among other groups. Moreover, the potential of LL-37 peptide to overcome the CRISPR/Cas9 delivery challenge was shown for the first time. Overall, our study suggests that the CRISPR/Cas9-mediated gene editing of pre-existing tumors is effective, specific and nontoxic, and the outlook for precise gene therapy in cancer patients is very bright.

Conserved multiepitope vaccine constructs: A potent HIV-1 therapeutic vaccine in clinical trials.

Despite the success of Antiretroviral Therapy (ART) in preventing HIV-1-associated clinical progression to AIDS, it is unable to eliminate the viral reservoirs and eradicate the HIV-1 infection. Therapeutic vaccination is an alternative approach to alter the HIV-1 infection course. It can induce effective HIV-1-specific immunity to control viremia and eliminate the need for lifelong ART. Immunological data from spontaneous HIV-1 controllers have shown that cross-reactive T-cell responses are the key immune mechanism in HIV-1 control. Directing these responses toward preferred HIV-1 epitopes is a promising strategy in therapeutic vaccine settings. Designing novel immunogens based on the HIV-1 conserved regions containing a wide range of critical T- and B-cell epitopes of the main viral antigens (conserved multiepitope approaches) supplies broad coverage of global diversity in HIV-1 strains and Human Leukocyte Antigen (HLA) alleles. It can also prevent immune induction to undesirable decoy epitopes theoretically. The efficacy of different novel HIV-1 immunogens based on the conserved and/or functional protective site of HIV-1 proteome has been evaluated in multiple clinical trials. Most of these immunogens were generally safe and able to induce potent HIV-1-specific immunity. However, despite these findings, several candidates have demonstrated limited efficacy in viral replication control. In this study, we used the PubMed and ClinicalTrial.gov databases to review the rationale of designing curative HIV-1 vaccine immunogens based on the conserved favorable site of the virus. Most of these studies evaluate the efficacy of vaccine candidates in combination with other therapeutics and/or with new formulations and immunization protocols. This review briefly describes the design of conserved multiepitope constructs and outlines the results of these vaccine candidates in the recent clinical pipeline.

Comparison of Adjuvant Effects of Montanide ISA-720 and Heat Shock Protein 27 in Increasing Immunostimulatory Properties of HIV-1 Nef-Vif Fusion Protein Construct.

INTRODUCTION: Effective T-cell-mediated immunity has emerged as an essential component of human immunodeficiency virus-1 (HIV-1) vaccination. Thus, inducing an immune response against HIV proteins such as Nef and Vif, two major accessory proteins with critical roles in HIV pathogenesis and immune evasion, may lead to an effective approach. AIM: Our goal is to evaluate and compare Montanide ISA-720 and heat shock protein 27 in increasing immunostimulatory properties of HIV-1 Nef-Vif fusion protein as a vaccine candidate. METHODS: In this study, the nef-vif fusion gene with and without the heat shock protein 27 (hsp27) gene was cloned in the prokaryotic pET24a (+) vector. Then, the recombinant Nef-Vif and Hsp27-Nef- Vif proteins were generated in the E. coli system. Finally, their immunostimulatory properties were evaluated in mice. Indeed, the potency of Hsp27 as an endogenous natural adjuvant was investigated to enhance HIV-1 Nef-Vif antigen-specific immunity compared to Montanide ISA-720 as a commercial adjuvant in protein-based immunization strategy. RESULTS: Our results approved the role of Hsp27 as an effective adjuvant in the stimulation of B- and T-cell immunity. The linkage of Hsp27 to antigen could elicit higher levels of IgG1, IgG2a, IFN-γ, IL- 5 and Granzyme B than antigen mixed with Montanide ISA-720. Moreover, the ratios of IFN-γ/IL-5 and IgG2a/IgG1 were significantly increased in groups receiving Nef-Vif protein + Montanide ISA- 720 and Hsp27-Nef-Vif protein indicating the direction of the immune response pathway toward strong Th1 response. These ratios were higher in the group receiving Hsp27-Nef-Vif protein than in the group receiving Nef-Vif protein + Montanide ISA-720. CONCLUSION: Our findings suggest that Hsp27 can be used as an effective adjuvant to enhance antigenspecific immune responses in HIV-1 infectious models for therapeutic vaccine development.

Application of Cell Penetrating Peptides as a Promising Drug Carrier to Combat Viral Infections.

Novel effective drugs or therapeutic vaccines have been already developed to eradicate viral infections. Some non-viral carriers have been used for effective drug delivery to a target cell or tissue. Among them, cell penetrating peptides (CPPs) attracted a special interest to enhance drug delivery into the cells with low toxicity. They were also applied to transfer peptide/protein-based and nucleic acids-based therapeutic vaccines against viral infections. CPPs-conjugated drugs or vaccines were investigated in several viral infections including poliovirus, Ebola, coronavirus, herpes simplex virus, human immunodeficiency virus, hepatitis B virus, hepatitis C virus, Japanese encephalitis virus, and influenza A virus. Some studies showed that the uptake of CPPs or CPPs-conjugated drugs can be performed through both non-endocytic and endocytic pathways. Despite high potential of CPPs for cargo delivery, there are some serious drawbacks such as non-tissue-specificity, instability, and suboptimal pharmacokinetics features that limit their clinical applications. At present, some solutions are utilized to improve the CPPs properties such as conjugation of CPPs with targeting moieties, the use of fusogenic lipids, generation of the proton sponge effect, etc. Up to now, no CPP or composition containing CPPs has been approved by the Food and Drug Administration (FDA) due to the lack of sufficient in vivo studies on stability, immunological assays, toxicity, and endosomal escape of CPPs. In this review, we briefly describe the properties, uptake mechanisms, advantages and disadvantages, and improvement of intracellular delivery, and bioavailability of cell penetrating peptides. Moreover, we focus on their application as an effective drug carrier to combat viral infections.

LL-37 antimicrobial peptide and heterologous prime-boost vaccination regimen significantly induce HIV-1 Nef-Vpr antigen- and virion-specific immune responses in mice.

OBJECTIVES: HIV infection still remains a leading cause of morbidity and mortality worldwide. The inability of highly-active antiretroviral therapy in HIV-1 eradication led to development of therapeutic vaccines. Exploiting effective immunogenic constructs and potent delivery systems are important to generate effective therapeutic vaccines, and overcome their poor membrane permeability. Among HIV-1 proteins, the Nef and Vpr proteins can be considered as antigen candidates in vaccine design. METHODS: In this study, the immunogenicity of Nef-Vpr antigen candidate in different regimens along with antimicrobial peptide LL-37 (as a DNA carrier) and Montanide 720 (as an adjuvant) was studied in mice. Moreover, the secretion of cytokines was assessed in virion-exposed mice lymphocytes in vitro. RESULTS: Our data indicated that groups immunized with the homologous protein + Montanide regimen (group 1), and also the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) could significantly generate strong immune responses as compared to groups immunized with the DNA constructs (groups 3 & 4). Moreover, immunization of mice with the homologous DNA + LL-37 regimen in low dose of DNA (5 µg) could induce higher immune responses than the homologous naked DNA regimen in high dose of DNA (50 µg) indicating the role of LL-37 as a cell penetrating peptide. Additionally, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen (group 2) induced significantly IFN-gamma secretion from virion-exposed lymphocytes in vitro. CONCLUSION: Generally, the use of LL-37 for DNA delivery, Montanide 720 as an adjuvant, and heterologous DNA prime/protein boost strategy could significantly increase IgG2a, IFN-gamma, and Granzyme B, and maintain cytokine secretion after exposure to virions. Indeed, the heterologous DNA + LL-37 prime/protein + Montanide boost regimen can be considered as a potent strategy for development of therapeutic HIV vaccines.

Lipid-Based Delivery Systems in Development of Genetic and Subunit Vaccines.

Lipidic carriers are composed of natural, synthetic, or physiological lipid/phospholipid materials. The flexibility of lipid-based delivery systems for transferring a variety of molecules such as immunomodulators, antigens, and drugs play a key role in design of effective vaccination and therapeutic strategies against infectious and non-infectious diseases. Genetic and subunit vaccines are two major groups of promising vaccines that have the potential for improving the protective potency against different diseases. These vaccine strategies rely greatly on delivery systems with various functions, including cargo protection, targeted delivery, high bioavailability, controlled release of antigens, selective induction of antigen-specific humoral or cellular immune responses, and low side effects. Lipidic carriers play a key role in local tissue distribution, retention, trafficking, uptake and processing by antigen-presenting cells. Moreover, lipid nanoparticles have successfully achieved to the clinic for the delivery of mRNA. Their broad potential was shown by the recent approval of COVID-19 mRNA vaccines. However, size, charge, architecture, and composition need to be characterized to develop a standard lipidic carrier. Regarding the major roles of lipid-based delivery systems in increasing the efficiency and safety of vaccine strategies against different diseases, this review concentrates on their recent advancements in preclinical and clinical trials.

Conserved multiepitope vaccine constructs: A potent HIV-1 therapeutic vaccine in clinical trials

Abstract Despite the success of Antiretroviral Therapy (ART) in preventing HIV-1-associated clinical progression to AIDS, it is unable to eliminate the viral reservoirs and eradicate the HIV-1 infection. Therapeutic vaccination is an alternative approach to alter the HIV-1 infection course. It can induce effective HIV-1-specific immunity to control viremia and eliminate the need for lifelong ART. Immunological data from spontaneous HIV-1 controllers have shown that cross-reactive T-cell responses are the key immune mechanism in HIV-1 control. Directing these responses toward preferred HIV-1 epitopes is a promising strategy in therapeutic vaccine settings. Designing novel immunogens based on the HIV-1 conserved regions containing a wide range of critical T- and B-cell epitopes of the main viral antigens (conserved multiepitope approaches) supplies broad coverage of global diversity in HIV-1 strains and Human Leukocyte Antigen (HLA) alleles. It can also prevent immune induction to undesirable decoy epitopes theoretically. The efficacy of different novel HIV-1 immunogens based on the conserved and/or functional protective site of HIV-1 proteome has been evaluated in multiple clinical trials. Most of these immunogens were generally safe and able to induce potent HIV-1-specific immunity. However, despite these findings, several candidates have demonstrated limited efficacy in viral replication control. In this study, we used the PubMed and ClinicalTrial.gov databases to review the rationale of designing curative HIV-1 vaccine immunogens based on the conserved favorable site of the virus. Most of these studies evaluate the efficacy of vaccine candidates in combination with other therapeutics and/or with new formulations and immunization protocols. This review briefly describes the design of conserved multiepitope constructs and outlines the results of these vaccine candidates in the recent clinical pipeline.

Different dendritic cells-based vaccine constructs influence HIV-1 antigen-specific immunological responses and cytokine generation in virion-exposed splenocytes.

In recent years, dendritic cells (DCs)-based vaccines have been developed to combat HIV-1 infection in preclinical and clinical trials. In this study, mice bone marrow cells-derived DCs were pulsed with the recombinant Nef, heat shock protein 27 (Hsp27) and Hsp27-Nef proteins, and also green fluorescent protein (GFP) as a positive control. Then, new platforms of DCs loaded with HIV-1 Nef and Hsp27-Nef proteins (i.e., DC prime/DC boost, DNA prime/DC boost, and DC prime/protein boost) were used to evaluate immune responses in BALB/c mice. Finally, the potency of splenocytes exposed to single-cycle replicable (SCR) HIV-1 virions was investigated to secret cytokines in vitro. Our data indicated that the recombinant Nef (∼30 kDa), Hsp27 (∼27 kDa), GFP (∼27 kDa), and Hsp27-Nef (∼53 kDa) proteins were greatly generated in E. coli. Moreover, the modified DCs with the recombinant proteins were prepared in large scale. The results of mice immunization showed the highest levels of antibodies, cytokines, and Granzyme B in heterologous DC prime/protein boost regimen using Hsp27-Nef antigen (DCHsp27-Nef prime/ protein Hsp27-Nef boost regimen). The levels of IFN-γ and IL-10 cytokines in splenocytes isolated from mice immunized with DCHsp27-Nef prime/ protein Hsp27-Nef boost regimen were higher than those in other regimens after exposure to SCR virions. These findings demonstrated the importance of Hsp27 as an adjuvant and heterologous DC prime/ protein boost regimen in improvement of immune responses. Indeed, DC Hsp27-Nef prime/ protein Hsp27-Nef boost regimen can be utilized as a promising candidate for HIV-1 vaccine development.

Cell penetrating peptide: A potent delivery system in vaccine development.

One of the main obstacles to most medication administrations (such as the vaccine constructs) is the cellular membrane's inadequate permeability, which reduces their efficiency. Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) are well-known as potent biological nanocarriers to overcome this natural barrier, and to deliver membrane-impermeable substances into cells. The physicochemical properties of CPPs, the attached cargo, concentration, and cell type substantially influence the internalization mechanism. Although the exact mechanism of cellular uptake and the following processing of CPPs are still uncertain; but however, they can facilitate intracellular transfer through both endocytic and non-endocytic pathways. Improved endosomal escape efficiency, selective cell targeting, and improved uptake, processing, and presentation of antigen by antigen-presenting cells (APCs) have been reported by CPPs. Different in vitro and in vivo investigations using CPP conjugates show their potential as therapeutic agents in various medical areas such as infectious and non-infectious disorders. Effective treatments for a variety of diseases may be provided by vaccines that can cooperatively stimulate T cell-mediated immunity (T helper cell activity or cytotoxic T cell function), and immunologic memory. Delivery of antigen epitopes to APCs, and generation of a potent immune response is essential for an efficacious vaccine that can be facilitated by CPPs. The current review describes the delivery of numerous vaccine components by various CPPs and their immunostimulatory properties.

Immunological investigation of a multiepitope peptide vaccine candidate based on main proteins of SARS-CoV-2 pathogen.

Multiepitope vaccines could induce multiantigenic immunity against large complex pathogens with different strain variants. Herein, the in silico, in vitro and in vivo studies were used to design and develop a novel candidate antigenic multiepitope vaccine against SARS-CoV-2 pathogen. The designed multiepitope construct targets the spike glycoprotein (S), membrane protein (M), and nucleocapsid phosphoprotein (N) of SARS-CoV-2 (i.e., the S-N-M construct). This construct contains the cytotoxic T lymphocyte (CTL)-, helper T lymphocyte (HTL)-, and linear B lymphocyte (LBL)-inducing epitopes. The multiepitope s-n-m fusion gene was subcloned in prokaryotic (pET24a) and eukaryotic (pcDNA3.1) expression vectors. Its expression was evaluated in mammalian cell line using LL37 cell penetrating peptide. Moreover, the recombinant multiepitope S-N-M peptide was produced in E. coli strain. Finally, mice were immunized using homologous and heterologous regimens for evaluation of immune responses. Our data indicated that the multiepitope S-N-M peptide construct combined with Montanide 720 in homologous regimen significantly stimulated total IgG, IgG2a, IFN-γ, TNF-α, IL-15, IL-21 and IL-6, and Granzyme B secretion as compared to other groups. Moreover, the pcDNA-s-n-m/ LL37 nanoparticles significantly induced higher immune responses than the naked DNA in both homologous and heterologous regimens. In general, our designed multiepitope vaccine construct can be considered as a vaccine candidate in SARS-CoV-2 infection model.