DNA Vaccination Strategy Research Articles

TRLS-05. A PILOT STUDY TO ASSESS THE SAFETY, FEASIBILITY, AND PRELIMINARY EFFICACY OF A NEOEPITOPE-BASED PERSONALIZED DNA VACCINE APPROACH IN PEDIATRIC PATIENTS WITH RECURRENT BRAIN TUMORS

Abstract BACKGROUND Pediatric brain tumors present a heterogeneous group of neoplasms with limited treatment options, especially at relapse. Hence, innovative therapeutic strategies are imperative. Immunotherapy, specifically immunogenomics, utilizing personalized vaccines based on tumor-specific neoantigens, has emerged as a promising option in other tumors. However, the feasibility of this approach in pediatric brain tumors remain largely unexplored. METHODS This single institution feasibility study focuses on sequencing analyses of pediatric brain tumors to identify mutant tumor-specific antigens. The study employs tumor/normal exome sequencing and cDNA-capture sequencing to identify and confirm mutant antigens, respectively. An epitope prediction algorithm prioritizes these antigens with subsequent confirmation of binding to HLA class I molecules. The neoantigen DNA vaccine strategy, utilizing a polyepitope DNA vaccine, is designed to target up to 20 prioritized antigens. The TDS-IM v2.0 device facilitates the administration of the vaccine. The primary objective of the study is to determine the feasibility of adjuvant personalized neoantigen DNA vaccine administration in patients with recurrent or refractory pediatric brain tumors. RESULTS We anticipate the identification of actionable neoantigens in pediatric brain tumors to support the potential efficacy of personalized DNA vaccines. The proposed sequencing strategies, epitope prediction algorithm, and in vitrobinding studies aim to establish a comprehensive methodology for neoantigen identification and prioritization. During the meeting, we will present the study background, rationale, detailed methodology, and design to inform the brain tumor community of this study. CONCLUSION This feasibility study is poised to provide essential insights into the development of personalized neoantigen DNA vaccines for recurrent or refractory pediatric brain tumors and aims to demonstrate safety, feasibility, and potential efficacy in targeting mutant tumor-specific antigens. The findings from this study will lay the groundwork for future clinical trials, evaluating the impact of personalized immunotherapy on progression-free and overall survival rates in this challenging patient population.

An innovative lab-scale production for a novel therapeutic DNA vaccine candidate against rheumatoid arthritis

BackgroundRecent therapeutic-plasmid DNA vaccine strategies for rheumatoid arthritis (RA) have significantly improved. Our pcDNA-CCOL2A1 vaccine is the most prominent and the first antigen-specific tolerising DNA vaccine with potent therapeutic and prophylactic effects compared with methotrexate (MTX), the current “gold standard” treatment for collagen-induced arthritis (CIA). This study developed a highly efficient, cost-effective, and easy-to-operate system for the lab-scale production of endotoxin-free supercoiled plasmids with high quality and high yield. Based on optimised fermentation culture, we obtained a high yield of pcDNA-CCOL2A1 vaccine by PEG/MgCl2 precipitation and TRION-114. We then established a method for quality control of the pcDNA-CCOL2A1 vaccine. Collagen-induced arthritis (CIA) model rats were subjected to intramuscular injection of the pcDNA-CCOL2A1 vaccine (300 μg/kg) to test its biological activity.ResultsAn average yield of 11.81 ± 1.03 mg purified supercoiled plasmid was obtained from 1 L of fermentation broth at 670.6 ± 57.42 mg/L, which was significantly higher than that obtained using anion exchange column chromatography and a commercial purification kit. Our supercoiled plasmid had high purity, biological activity, and yield, conforming to the international guidelines for DNA vaccines.ConclusionThe proposed innovative downstream process for the pcDNA-CCOL2A1 vaccine can not only provide a large-scale high-quality supercoiled plasmid DNA for preclinical research but also facilitate further pilot-scale and even industrial-scale production of pcDNA-CCOL2A1 vaccine.

A follow-up study: 6-year cART-free virologic control of rhesus macaques after PD-1-based DNA vaccination against pathogenic SHIVSF162P3CN challenge.

An effective vaccine for combination antiretroviral therapy (cART)-free virologic control of HIV-1 by enhancing antigen-specific effector-memory CD8+ T lymphocytes could be useful in controlling the AIDS pandemic. Previously, we demonstrated that a programmed death-1 (PD-1)-based DNA vaccine, pRhPD1-p27, was effective in inducing Gag-p27-specific effector-memory CD8+ T cells for viremia suppression in rhesus macaques infected with pathogenic SHIVSF162P3CN for 2 years. In this follow-up study, we report on two pRhPD1-p27-vaccinated and SHIVSF162P3CN-infected macaques that have remained alive to date with undetectable viremia levels and low proviral loads over 6 years in the absence of cART, achieving a state of sustained virologic control. Polyfunctional effector-memory Gag-p27-specific T cells were maintained in these macaques. Moreover, several T cell epitopes found at 6 years post-viral challenge were identical to those induced during the vaccination phase, indicating sustained vaccine-induced memory T cell responses. The viral challenge resulted in de novo Nef-specific T cell responses, which were also maintained. These Gag-p27- and Nef-specific T cell responses were stronger than those in some SHIVSF162P3CN-infected macaques that showed viremia control after experimental therapy using a tandem bispecific neutralizing antibody. Our findings indicate that the PD-1-based DNA vaccine strategy holds promise as a clinical immunotherapy for long-term HIV-1 suppression.IMPORTANCEEfficient strategies for HIV-1 cART-free virologic control are critical for ending the AIDS pandemic. The essential role of effector-memory CD8+ T cells in controlling viremia and eliminating virus-infected cells has made them a promising target for vaccine development. It has been previously reported that PD-1-based DNA vaccination was effective in inducing polyfunctional effector-memory CD8+ T cells for AIDS virus control for 2 years in rhesus monkeys. This follow-up study extends the findings and shows that a viremia-free period of over 6 years was detected in two monkeys immunized with PD-1-based DNA vaccine against pathogenic SHIVSF162P3CN infection in the absence of antiretroviral therapy. Long-term vaccine-induced memory T cell responses were detected. Our results warrant the clinical trials of PD-1-based DNA vaccines for achieving HIV-1 cART-free virologic control used either alone or in combination with other biomedical interventions.

Title: Immunotherapy; a ground-breaking remedy for spinal cord injury with stumbling blocks: An overview.

Spinal cord injury (SCI) is a debilitating disorder with no known standard and effective treatment. Despite its ability to exacerbate SCI sequel by accelerating auto-reactive immune cells, an immune response is also considered essential to the healing process. Therefore, immunotherapeutic strategies targeting spinal cord injuries may benefit from the dual nature of immune responses. An increasing body of research suggests that immunization against myelin inhibitors can promote axon remyelination after SCI. However, despite advancements in our understanding of neuroimmune responses, immunoregulation-based therapeutic strategies have yet to receive widespread acceptance. Therefore, it is a prerequisite to enhance the understanding of immune regulation to ensure the safety and efficacy of immunotherapeutic treatments. The objective of the present study was to provide an overview of previous studies regarding the advantages and limitations of immunotherapeutic strategies for functional recovery after spinal cord injury, especially in light of limiting factors related to DNA and cell-based vaccination strategies by providing a novel prospect to lay the foundation for future studies that will help devise a safe and effective treatment for spinal cord injury.

An intranasal stringent response vaccine targeting dendritic cells as a novel adjunctive therapy against tuberculosis.

Lengthy tuberculosis (TB) treatment is required to overcome the ability of a subpopulation of persistent Mycobacterium tuberculosis (Mtb) to remain in a non-replicating, antibiotic-tolerant state characterized by metabolic remodeling, including induction of the RelMtb-mediated stringent response. We developed a novel therapeutic DNA vaccine containing a fusion of the relMtb gene with the gene encoding the immature dendritic cell-targeting chemokine, MIP-3α/CCL20. To augment mucosal immune responses, intranasal delivery was also evaluated. We found that intramuscular delivery of the MIP-3α/relMtb (fusion) vaccine or intranasal delivery of the relMtb (non-fusion) vaccine potentiate isoniazid activity more than intramuscular delivery of the DNA vaccine expressing relMtb alone in a chronic TB mouse model (absolute reduction of Mtb burden: 0.63 log10 and 0.5 log10 colony-forming units, respectively; P=0.0002 and P=0.0052), inducing pronounced Mtb-protective immune signatures. The combined approach involving intranasal delivery of the DNA MIP-3α/relMtb fusion vaccine demonstrated the greatest mycobactericidal activity together with isoniazid when compared to each approach alone (absolute reduction of Mtb burden: 1.13 log10, when compared to the intramuscular vaccine targeting relMtb alone; P<0.0001), as well as robust systemic and local Th1 and Th17 responses. This DNA vaccination strategy may be a promising adjunctive approach combined with standard therapy to shorten curative TB treatment, and also serves as proof of concept for treating other chronic bacterial infections.

Fast DNA Vaccination Strategy Elicits a Stronger Immune Response Dependent on CD8+CD11c+ Cell Accumulation.

Conventional DNA vaccine strategies usually employ a regimen of immunizations at 2-week or longer intervals to induce effective memory cell-dependent immune responses. Clinical cancer treatment requires a faster immunization strategy to contend with tumor progression. In this study, a novel fast immunization strategy was established, wherein a DNA vaccine was intramuscularly administered on days 0, 2, and 5 in a murine lung cancer model. Effector cells peaked 7 to 10 days after the last vaccination. Compared with traditional 2-week-interval immunization strategies, antigen-specific cytolysis and INF-γ secretion were significantly enhanced under the fast vaccination approach. As a result, the rapidly administered DNA vaccine elicited stronger and more prompt antitumor effects. The probable underlying mechanism of fast immunization was the accumulation of CD8+CD11c+ antigen-presenting cells at the injection site, which enhanced subsequent antigen presentation. In conclusion, the fast DNA vaccination strategy shortened vaccination time to 5 days and elicited a stronger antitumor immune response.

An Update on the HIV DNA Vaccine Strategy

In 2020, the global prevalence of human immunodeficiency virus (HIV) infection was estimated to be 38 million, and a total of 690,000 people died from acquired immunodeficiency syndrome (AIDS)–related complications. Notably, around 12.6 million people living with HIIV/AIDS did not have access to life-saving treatment. The advent of the highly active antiretroviral therapy (HAART) in the mid-1990s remarkably enhanced the life expectancy of people living with HIV/AIDS as a result of improved immune functions. However, HAART has several drawbacks, especially when it is not used properly, including a high risk for the development of drug resistance, as well as undesirable side effects such as lipodystrophy and endocrine dysfunctions, which result in HAART intolerability. HAART is also not curative. Furthermore, new HIV infections continue to occur globally at a high rate, with an estimated 1.7 million new infections occurring in 2018 alone. Therefore, there is still an urgent need for an affordable, effective, and readily available preventive vaccine against HIV/AIDS. Despite this urgent need, however, progress toward an effective HIV vaccine has been modest over the last four decades. Reasons for this slow progress are mainly associated with the unique aspects of HIV itself and its ability to rapidly mutate, targeting immune cells and escape host immune responses. Several approaches to an HIV vaccine have been undertaken. However, this review will mainly discuss progress made, including the pre-clinical and clinical trials involving vector-based HIV DNA vaccines and the use of integrating lentiviral vectors in HIV vaccine development. We concluded by recommending particularly the use of integrase-defective lentiviral vectors, owing to their safety profiles, as one of the promising vectors in HIV DNA vaccine strategies both for prophylactic and therapeutic HIV vaccines.

Brief review of the mRNA vaccines COVID-19.

The global COVID-19 pandemic continues to claim victims worldwide, representing a health and socioeconomic challenge with few precedents in human history. The therapeutic agents used to treat COVID-19 infection are mostly aimed at avoiding the most serious complications and organ damage that in a percentage of cases can be fatal. Recently, the first COVID-19 vaccines have been authorised, starting a massive vaccination campaign worldwide. The COVID-19 vaccines authorized or under testing use different methods of action such as mRNA, DNA vaccine, viral vector, protein subunit, and virus inactivated immunization strategies. The mRNA vaccines are the first authorised vaccines with this new method of action, initiating a new era of preventive medicine. To date, data indicate that they may be effective against the newly identified SARS-CoV-2 variants. Short-term studies indicated an acceptable safety profile but long-term immunity as well as safety is not yet available. In this article, we briefly describe mRNA vaccines and highlight some aspects yet to be fully defined.

Peptide: MHC-based DNA vaccination strategy to activate natural killer cells by targeting killer cell immunoglobulin-like receptors

BackgroundNatural killer (NK) cells are increasingly being recognized as agents for cancer immunotherapy. The killer cell immunoglobulin-like receptors (KIRs) are expressed by NK cells and are immunogenetic determinants of the...

In Silico Prediction of T and B Cell Epitopes of SAG1-Related Sequence 3 (SRS3) Gene for Developing Toxoplasma gondii Vaccine

: Toxoplasmosis is a worldwide infection that can lead to serious problems in immune-compromised individuals and fetuses. A DNA vaccine strategy would be an ideal tool against Toxoplasma gondii. One of the necessary measures to provide an effective vaccine is the selection of proteins with high antigenicity. The SAG1-related sequence 3 (SRS3) protein is a major surface antigen in T. gondii that can be used as a vaccine candidate. In the present study, bioinformatics and computational methods were utilized to predict protein characteristics, as well as secondary and tertiary structures. The in silico approach is highly suited to analyze, design, and evaluate DNA vaccine strategies. Hence, in silico prediction was used to identify B and T cell epitopes and compare the antigenicity of SRS3 and other candidate genes of Toxoplasma previously applied in the production of vaccines. The results of the analysis theoretically showed that SRS3 has multiple epitopes with high antigenicity, proposing that SRS3 is a promising immunogenic candidate for the development of DNA vaccines against toxoplasmosis.

Enhanced recombinant C-terminal domain of gli2 gene expression can improve wound healing through promoting cdc25b and N-Myc genes expression

Wound is an important cause of morbidity and mortality in population because it can be caused by many factors like events, illness and surgery. Identification of wound healing factors and their role can be useful for effective treatment. GLI2 as an important activator of SHH signaling pathway should be further investigated. In this study we focused on the effect of a recombinant derivative of GLI2 gene on the progression of wound healing and expression of cdc25a and Myc genes as two transcription factors involved in wound healing pathways.A fragment containing coding sequence of functional domains of C-terminal of GLI2 protein was cloned in pcDNA3.1+ plasmid. A 1 cm diameter circular area wound up on the back of the one group of wistar albino rats. In the next step, purified recombinant plasmid containing GLI2 C-terminal domains coding sequence, was injected subcutaneously around the wound similar to what we do about the DNA vaccine strategy. Wound area was following during 7 days and all results was documented by digital pictures. Peripheral tissue of the wounds was dissected and studied for cdc25a and N-Myc genes expression real-time RT-PCR analysis. Total of wound scare was dissected after 7 days since injection. These biopsies were subjected to RNA extraction and Real-Time PCR analysis for expression analysis of Gli2, N-myc and cdc25b genes. In addition, a piece of every biopsy was analyzed by immunohistochemistry method for detection and analysis of gli2 protein.Our results showed a significant improvement in wound healing treatment by recombinant injected pcDNA3.1/GLI compared with the control group. Also, the affected tissue of rats that express recombinant gli2, had significant increasing of N-myc and cdc25b gene expression, which leads to acceleration of cell proliferation during wound healing.GLI2 protein is the major activator of SHH pathway and this signaling route has a vital role in renewing and repair of tissues.GLI2 protein can be consider as a potent factor in wound healing. Especially GLI2 as a transcription factor can regulates the expression of many proteins that play important role in healing process. Our results support the function of some of GLI motives in gene expression separately. Overexpression of N-Myc and cdc25b genes compared with the control group, can indirectly explain the function mechanism of GLI2 in the tissue repair.

DNA nanovaccines prepared using LemA antigen protect Golden Syrian hamsters against Leptospira lethal infection.

BACKGROUND Nanoparticles (NPs) are viable candidates as carriers of exogenous materials into cells via transfection and can be used in the DNA vaccination strategy against leptospirosis.OBJECTIVES We evaluated the efficiency of halloysite clay nanotubes (HNTs) and amine-functionalised multi-walled carbon nanotubes (NH2-MWCNTs) in facilitating recombinant LemA antigen (rLemA) expression and protecting Golden Syrian hamsters (Mesocricetus auratus) against Leptospira interrogans lethal infection.METHODS An indirect immunofluorescent technique was used to investigate the potency of HNTs and NH2-MWCNTs in enhancing the transfection and expression efficiency of the DNA vaccine in Chinese hamster ovary (CHO) cells. Hamsters were immunised with two doses of vaccines HNT-pTARGET/lemA, NH2-MWCNTs-pTARGET/lemA, pTARGET/lemA, and empty pTARGET (control), and the efficacy was determined in terms of humoral immune response and protection against a lethal challenge.FINDINGS rLemA DNA vaccines carried by NPs were able to transfect CHO cells effectively, inducing IgG immune response in hamsters (p < 0.05), and did not exhibit cytotoxic effects. Furthermore, 83.3% of the hamsters immunised with NH2-MWCNTs-pTARGET/lemA were protected against the lethal challenge (p < 0.01), and 66.7% of hamsters immunised with HNT-pTARGET/lemA survived (p < 0.05).MAIN CONCLUSIONS NH2-MWCNTs and HNTs can act as antigen carriers for mammalian cells and are suitable for DNA nanovaccine delivery.

Immune Responses Induced by pVAX/TgERK7 against Toxoplasma gondii Infection in BALB/c Mice

Toxoplasma gondii can infect all the warm-blooded vertebrates and cause serious toxoplasmosis. Extracellular signal-regulated kinase 7 in T. gondii (TgERK7) mediated the proliferation of this parasite may be a potential vaccine candidate. Thus, immune responses induced by TgERK7 were investigated in this study using a DNA vaccine strategy. pVAX/TgERK7 plasmid was constructed and used to immunize BALB/c mice for three times with two-week intervals. The challenge and the investigation of humoral and cellular immune responses were performed at two weeks post the last immunization, and the survival times of the infected mice were daily recorded until all of them were dead. The innate immune response with higher concentrations of IFN-γ, TNF-α, IL2 and IL12p70 in sera (P < 0.05), and the adaptive immune responses were evoked by the DNA immunizations, including specific antibody, lymphocyte proliferation, and the CD3e+CD4+ and CD3e+CD8a+ T cell-mediated response effects. Interestingly, no significant difference was detected in their survival times among all the experimental groups of mice that were challenged with GT1 tachyzoites or PRU cysts (P>0.05). The successive immunizations with pVAX/TgERK7 can provoke the innate and adaptive immune responses of BALB/c mice, whereas the DNA vaccine-induced immunological efficacy is not sufficient for complete protection the host against T. gondii infection.

A Hepatitis C Virus DNA Vaccine Encoding a Secreted, Oligomerized Form of Envelope Proteins Is Highly Immunogenic and Elicits Neutralizing Antibodies in Vaccinated Mice.

Hepatitis C virus (HCV) persistently infects approximately 71 million people globally. To prevent infection a vaccine which elicits neutralizing antibodies against the virus envelope proteins (E1/E2) which are required for entry into host cells is desirable. DNA vaccines are cost-effective to manufacture globally and despite recent landmark studies highlighting the therapeutic efficacy of DNA vaccines in humans against cervical cancer, DNA vaccines encoding E1/E2 developed thus far are poorly immunogenic. We now report a novel and highly immunogenic DNA vaccination strategy that incorporates secreted E1 and E2 (sE1 and sE2) into oligomers by fusion with the oligomerization domain of the C4b-binding protein, IMX313P. The FDA approved plasmid, pVax, was used to encode sE1, sE2, or sE1E2 with or without IMX313P, and intradermal prime-boost vaccination studies in BALB/c mice showed that vaccines encoding IMX313P were the most effective in eliciting humoral and cell-mediated immunity against the envelope proteins. Further boosting with recombinant E1E2 proteins but not DNA nor virus-like particles (VLPs) expressing E1E2 increased the immunogenicity of the DNA prime-boost regimen. Nevertheless, the antibodies generated by the homologous DNA prime-boost vaccinations more effectively inhibited the binding of VLPs to target cells and neutralized transduction with HCV pseudoparticles (HCVpp) derived from different genotypes including genotypes 1, 2, 3, 4, 5, and 6. This report provides the first evidence that IMX313P can be used as an adjuvant for E1/E2-based DNA vaccines and represents a translatable approach for the development of a HCV DNA vaccine.

A DNA Vaccine Encoding the Gn Ectodomain of Rift Valley Fever Virus Protects Mice via a Humoral Response Decreased by DEC205 Targeting.

The Rift Valley fever virus (RVFV) is responsible for a serious mosquito-borne viral disease in humans and ruminants. The development of a new and safer vaccine is urgently needed due to the risk of introduction of this arbovirus into RVFV-free continents. We recently showed that a DNA vaccine encoding eGn, the ectodomain of the RVFV Gn glycoprotein, conferred a substantial protection in the sheep natural host and that the anti-eGn IgG levels correlated to protection. Addressing eGn to DEC205 reduced the protective efficacy while decreasing the antibody and increasing the IFNγ T cell responses in sheep. In order to get further insight into the involved mechanisms, we evaluated our eGn-encoding DNA vaccine strategy in the reference mouse species. A DNA vaccine encoding eGn induced full clinical protection in mice and the passive transfer of immune serum was protective. This further supports that antibodies, although non-neutralizing in vitro, are instrumental in the protection against RVFV. Addressing eGn to DEC205 was also detrimental to protection in mice, and in this species, both the antibody and the IFNγ T cell responses were strongly decreased. Conversely when using a plasmid encoding a different antigen, i.e., mCherry, DEC205 targeting promoted the antibody response. Altogether our results show that the outcome of targeting antigens to DEC205 depends on the species and on the fused antigen and is not favorable in the case of eGn. In addition, we bring evidences that eGn in itself is a pertinent antigen to be included in a DNA vaccine and that next developments should aim at promoting the anti-eGn antibody response.

Intra-muscular and oral vaccination using a Koi Herpesvirus ORF25 DNA vaccine does not confer protection in common carp (Cyprinus carpio L.)

Koi Herpes Virus (KHV or Cyprinid Herpesvirus 3, CyHV-3) is among the most threatening pathogens affecting common carp production as well as the highly valuable ornamental koi carp. To date, no effective commercial vaccine is available for worldwide use. A previous study reported that three intramuscular injections with an ORF25-based DNA vaccine, led to the generation of neutralizing antibodies and conferred significant protection against an intraperitoneal challenge with KHV. In the present study, we set out to optimize an ORF25-based DNA vaccination protocol that required fewer injections and would confer protection upon a challenge that better resembled the natural route of infection. To this end, ORF25 was cloned in pcDNA3 either as a soluble protein or as a full-length transmembrane GFP-fusion protein. We tested our ORF25-based DNA vaccines in multiple vaccination trials using different doses, vaccination routes (i.m. injection and oral gavage) and challenge methods (bath and cohabitation). Furthermore, we analysed local and systemic responses to the i.m. injected DNA vaccine through histological and RT-qPCR analysis. We observed a strong protection when fish received three injections of either of the two DNA vaccines. However, this protection was observed only after bath challenge and not after cohabitation challenge. Furthermore, protection was insufficient when fish received one injection only, or received the plasmid orally. The importance of choosing a challenge model that best reflects the natural route of infection and the possibility to include additional antigens in future DNA vaccination strategies against KHV will be discussed.

IRES-based co-expression of influenza virus conserved genes can promote synergistic antiviral effects both in vitro and in vivo.

Vaccination is the most effective method for the prevention of influenza virus infection. Currently used influenza vaccines that target the highly polymorphic viral surface antigens can provide protection when well matched with circulating virus strains. Antigenic drift or cyclically occurring pandemics may hamper the efficacy of these vaccines, which are chosen prior to each flu season. Therefore, a universal vaccine, designed to induce broadly cross-protective immunity against the highly conserved internal antigens M1 and nucleoprotein could provide durable protection against various influenza virus subtypes, and it could also reduce the impact of pandemic influenza, which occurs less frequently. Here, we describe a new influenza vaccine candidate in which two highly conserved antigens, nucleoprotein (NP) and matrix (M1), are simultaneously expressed from a bicistronic vector termed pIRESM1/NP. Mice were immunized intradermally four times with the pIRESM1/NP construct. The protection efficacy of the gene-based vaccine was assessed by IFN-γ and Granzyme B ELISpot assays, follow-up observation of weight loss, and survival rates of the mice groups against lethal challenges with influenza A virus subtypes H1N1 and H5N1. The group that received pIRESM1/NP showed full protection against disease following lethal challenge with H1N1 and H5N1. This group also generated significantly higher host immune cellular responses than the other groups. These results demonstrate that a DNA vaccine strategy based on co-expression of the M1 and NP proteins could provide an effective way to control influenza virus infection.

Hantavirus Gc induces long-term immune protection via LAMP-targeting DNA vaccine strategy

Hemorrhagic fever with renal syndrome (HFRS) occurs widely throughout Eurasia. Unfortunately, there is no effective treatment, and prophylaxis remains the best option against the major pathogenic agent, hantaan virus (HTNV), which is an Old World hantavirus. However, the absence of cellular immune responses and immunological memory hampers acceptance of the current inactivated HFRS vaccine. Previous studies revealed that a lysosome-associated membrane protein 1 (LAMP1)-targeting strategy involving a DNA vaccine based on the HTNV glycoprotein Gn successfully conferred long-term immunity, and indicated that further research on Gc, another HTNV antigen, was warranted. Plasmids encoding Gc and lysosome-targeted Gc, designated pVAX-Gc and pVAX-LAMP/Gc, respectively, were constructed. Proteins of interest were identified by fluorescence microscopy following cell line transfection. Five groups of 20 female BALB/c mice were subjected to the following inoculations: inactivated HTNV vaccine, pVAX-LAMP/Gc, pVAX-Gc, and, as the negative controls, pVAX-LAMP or the blank vector pVAX1. Humoral and cellular immunity were assessed by enzyme-linked immunosorbent assays (ELISAs) and 15-mer peptide enzyme-linked immunospot (ELISpot) epitope mapping assays. Repeated immunization with pVAX-LAMP/Gc enhanced adaptive immune responses, as demonstrated by the specific and neutralizing antibody titers and increased IFN-γ production. The inactivated vaccine induced a comparable humoral reaction, but the negative controls only elicited insignificant responses. Using a mouse model of HTNV challenge, the in vivo protection conferred by the inactivated vaccine and Gc-based constructs (with/without LAMP recombination) was confirmed. Evidence of pan-epitope reactions highlighted the long-term cellular response to the LAMP-targeting strategy, and histological observations indicated the safety of the LAMP-targeting vaccines. The long-term protective immune responses induced by pVAX-LAMP/Gc may be due to the advantage afforded by lysosomal targeting after exogenous antigen processing initiation and major histocompatibility complex (MHC) class II antigen presentation trafficking. MHC II-restricted antigen recognition effectively primes HTNV-specific CD4+ T-cells, leading to the promotion of significant immune responses and immunological memory. An epitope-spreading phenomenon was observed, which mirrors the previous result from the Gn study, in which the dominant IFN-γ-responsive hot-spot epitopes were shared between HLA-II and H2d. Importantly, the pan-epitope reaction to Gc indicated that Gc should be with potential for use in further hantavirus DNA vaccine investigations.

Increase in Incidence of Clostridium Difficile infection in aged models and effective DNA vaccine strategies for the prevention of disease

Abstract Clostridium difficile–associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases in industrialized nations and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Key toxin-neutralizing epitopes have been discovered within the carboxy-terminal receptor-binding domain (RBD) of the toxins, which has generated interest in developing the RBD as a vaccine target. The elderly mount an impaired humoral response to C. difficile toxins leading to more severe and recurrent disease; however, it has yet to be determined what underlying mechanisms lead to lower anti-toxin titers, thus higher incidence of severe and recurrent disease. While numerous platforms have been studied, very few data describe the potential of DNA vaccination against CDAD. We immunized C57BL/6 mice with plasmids encoding the RBD from TcdA and TcdB intramuscularly followed by in vivo electroporation. Vaccination induced significant levels of anti-RBD antibodies within the serum and feces that could neutralize toxins in an in vitro cytotoxicity assay. Aged animals had lower endpoint and anti-toxin neutralization titers, but aged mice still elicited functional antibody post vaccination. Both young and aged immunized mice were protected following orogastric challenge with strains of C. difficile spores that were homologous (VPI 10463; n=10/10) and heterologous (hypervirulent UK1; n=4/8) strains to our vaccine antigens. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical young adult and aged models of acute toxin-associated disease.

In vitro evaluation of a polytope DNA construct as a novel DNA vaccine strategy against human cytomegalovirus-associated diseases.

Human cytomegalovirus (HCMV) establishes severe disease in fetus, newborn and immunocompromised individuals. Polytope DNA vaccine strategy allows us to choose conserved and immunodominant epitopes from different antigens that can stimulate cellular and humoral immune responses simultaneously. In this study, a synthetic chimeric gene fragment was subcloned in to DNA vaccine vector pcDNA3.1+. The recombinant vector was transferred in to suitable eukaryotic cell line HEK 293T and the expression level of polytope construct from HEK 293T-infected cells was determined by western blot. These results show that there was no mutantion in target segment and recombinant vector showed significant levels of expression. Base on these results, using a proper procedure for design can cause expression and stability of polytope peptide.