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Background: Hantaviral diseases can have a high case fatality rate within the absence of broadly effective antiviral treatments or vaccines. We developed a DNA vaccine targeting the Hantavirus glycoprotein N-terminal (Gn) to major histocompatibility complex class II compartment by fusing the antigen with lysosome-associated membrane protein 1 (LAMP1), which altered antigen presenting pathway and activated the CD4+ T cells., Methods: The segments of Gn and LAMP1 were cloned into vector pVAX1, and recombinant plasmid was constructed by inserting Gn sequence into LAMP1, between luminal and the transmembrane/cytoplasmic domains. Subsequently, the protein expression was identified through immunoprecipitation, western blot and Immunofluorescent assay. Adaptive immune responses were assessed by the presence of specific and neutralizing antibodies, interferon (ELISpot results, and cytotoxic T-lymphocyte (CTL) cytotoxicity. Epitope mapping was performed to study the T-cell epitopes. Protective immunity in vivo was evaluated using a novel HTNV-challenging model, and safety evaluation was based on histological and behavioral observations., Results: Native or LAMP1 targeting HTNV Gn was successfully identified. Humoral immune responses were enhanced, featuring with satisfying titers of specific and neutralizing antibody production. The boosted activities of IFN-γ and CTL cytotoxicity witnessed enhanced cellular immune responses. Effective protection against HTNV in vivo was conferred in all three vaccine groups by the challenge model. Safety was confirmed and one dominant T-cell epitope screened from immunized mice overlapped the specific T-cell hot spot in HFRS patients., Conclusion: LAMP1 targeting strategy successfully enhanced the efficacy of HTNV Gn-based vaccine, which is highly immunogenic and safe, showing promise for immunoprophylaxis against HFRS. Further investigations are warranted in the future., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

HIV reverse transcriptase (RT) can be considered as a target and an instrument of immunotherapy aimed at limiting the emergence and spread of drug-resistant HIV. The chimeric genes coding for the wild-type and multi-drug-resistant RT (RT1.14) fused to lysosome-associated membrane protein 1 (LAMP-1) were injected intramuscularly into BALB/c mice. The immune response was assessed by ELISpot, cytokine ELISA intracellular IFN-gamma staining, and antibody ELISA. The genes for RT- and RT1.14-LAMP fusions (RT-LAMP and RT1.14-LAMP) were immunogenic generating a mixed Th1/Th2-profile of immune response, while the wild-type RT gene induced only weak immune response. Specific secretion of Th1-cytokines increased with increasing level of RT modification: RT

Rabies is progressive fatal encephalitis. WHO estimates 55,000 rabies deaths and more than 10 million PEP every year world-wide. A variety of cell-culture derived vaccines are available for prophylaxis against rabies. However, their high cost restricts their usage in developing countries, where such cases are most often encountered. This is driving the quest for newer vaccine formulations; DNA vaccines being most promising amongst them. Here, we explored strategies of antigen trafficking to various cellular compartments aiming at improving both humoral and cellular immunity. These strategies include use of signal sequences namely Tissue Plasminogen Activator (TPA), Ubiquitin (UQ) and Lysosomal-Associated Membrane Protein-1 (LAMP-1). TPA, LAMP-1 and their combination were aimed at enhancing the CD4(+) T cell and antibody response. In contrast, the UQ tag was utilized for enhancing CD8(+) response. The potency of modified DNA vaccines assessed by total antibody response, antibody isotypes, cytokine profile, neutralizing antibody titer and protection conferred against in vivo challenge; was enhanced in comparison to native unmodified vaccine, but the response elicited did not pertain to the type of target sequence and the directed arm of immunity. Interestingly, the DNA vaccines that had been designed to generate different type of immune responses yielded in effect similar response. In conclusion, our data indicate that the directing target sequence is not the exclusive deciding factor for type and extent of immune response elicited and emphasizes on the antigen dependence of immune enhancement strategies.

To determine the immunogenicity and protective efficacy of the Mycobacterium tuberculosis 10 kD culture filtrate protein (CFP10), and to evaluate strategies that enhance local immunity, we used C57Bl/6 DR4 mice that were transgenic for human HLA DRB1 0401, because CFP10 contains epitopes for DRB1 0401 but not for C57Bl/6 mice. Intramuscular immunization with a DNA vaccine encoding CFP10 elicited production of IFN-gamma by systemic CD4+ T cells, and one intravenous dose of the CFP10-based DNA vaccine coated with polyethylenimine (PEI) stimulated IFN-gamma production by lung CD4+ cells and reduced the pulmonary bacillary burden. We conclude that CFP10 is a potential vaccine candidate and that coating vaccines with PEI enhances local protective immunity to tuberculosis

Peptide-based vaccines, one of several anti-tumor immunization strategies currently under investigation, can elicit both MHC Class I-restricted (CD8(+)) and Class II-restricted (CD4(+)) responses. However, the need to identify specific T-cell epitopes in the context of MHC alleles has hampered the application of this approach. We have tested overlapping synthetic peptides (OSP) representing a tumor antigen as a novel approach that bypasses the need for epitope mapping, since OSP contain all possible epitopes for both CD8(+) and CD4(+) T cells. Here we report that vaccination of inbred and outbred mice with OSP representing tumor protein D52 (TPD52-OSP), a potential tumor antigen target for immunotherapy against breast, prostate, and ovarian cancer, was safe and induced specific CD8(+) and CD4(+) T-cell responses, as demonstrated by development of specific cytotoxic T cell (CTL) activity, proliferative responses, interferon (IFN)-gamma production and CD107a/b expression in all mice tested. In addition, TPD52-OSP-vaccinated BALB/c mice were challenged with TS/A breast carcinoma cells expressing endogenous TPD52; significant survival benefits were noted in vaccine recipients compared to unvaccinated controls (p<0.001). Our proof-of-concept data demonstrate the safety and efficacy of peptide library-based cancer vaccines that obviates the need to identify epitopes or MHC backgrounds of the vaccinees. We show that an OSP vaccination approach can assist in the disruption of self-tolerance and conclude that our approach may hold promise for immunoprevention of early-stage cancers in a general population.

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4(+) and CD8(+) cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection.