Cytolysin A-mediated protein exportation efficiency and its role in enhancing the fitness of live recombinant Salmonella Typhi vaccine strain.

The genetic fusion of cytolysin A (clyA) to heterologous antigen expressed in live Salmonella vector demonstrated efficient translocation into periplasmic space and extracellular medium. Accumulating evidence has shown that clyA-mediated antigen delivery improved growth fitness and enhanced immunogenicity of live vector vaccine, but the factors influencing this protein exportation has not been investigated. In this study, Toxoplasma gondii antigen fused at C-terminal of clyA protein was expressed in live S. Typhi vector via both plasmid and chromosomal-based expressions. The bivalent strains showed comparable growth rates as monovalent strains, but in varies antigen exportation efficiency. ClyA-fusion antigen with positive charges was translocated to the extracellular spaces, whereas those with negative charges were retained in the cytoplasm. Furthermore, excessive cellular resources expenditure on antigen expression, especially antigen with larger size, could limit the clyA-fusion antigen exportation, resulting in undesirable metabolic burden that eventually affects the growth fitness. Altogether, the present work indicates potential linkage of factors mainly on antigen properties and expression platforms that may affect clyA-mediated antigen delivery to enhance the growth fitness of live vector strain.

Efficacy of mRNA, adenoviral vector, and perfusion protein COVID-19 vaccines.

Coronavirus disease 2019 (COVID-19) has a devastating impact on global populations triggered by a highly infectious viral sickness, produced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The third major cause of mortality in the United States, following heart disease and cancer in 2020, was undoubtedly COVID-19. The centers for disease control and prevention (CDC) and the world health organization (WHO) separately developed a categorization system for differentiating new strains of SARS-CoV-2 into variants of concern (VoCs) and variants of interest (VoIs) with the continuing development of various strains SARS-CoV-2. By December 2021, five of the SARS-CoV-2 VoCs were discovered from the onset of the pandemic depending on the latest epidemiologic report by the WHO: Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529). Mutations in the receptor-binding domain (RBD) and n-terminal domain (NTD) have been found throughout all five identified VoCs. All strains other than the delta mutant are often found with the N501Y mutation situated on the RBD, resulting in higher binding between the spike protein and angiotensin-converting enzyme 2 (ACE2) receptors, enhanced viral adhesion, and following the entrance to host cells. The introduction of these new strains of SRAS-CoV-2 is likely to overcome the remarkable achievements gained in restricting this viral disease to the point where it is presented with remarkable vaccine developments against COVID-19 and strong worldwide mass immunization initiatives. Throughout this literature review, the effectiveness of current COVID-19 vaccines for managing and prohibiting SARS-CoV-2 strains is thoroughly described.

Anti-SARS-CoV-2 antibodies elicited by COVID-19 mRNA vaccine exhibit a unique glycosylation pattern.

Messenger RNA-based vaccines against COVID-19 induce a robust anti-SARS-CoV-2 antibody response with potent viral neutralization activity. Antibody effector functions are determined by their constant region subclasses and by their glycosylation patterns, but their role in vaccine efficacy is unclear. Moreover, whether vaccination induces antibodies similar to those in patients with COVID-19 remains unknown. We analyze BNT162b2 vaccine-induced IgG subclass distribution and Fc glycosylation patterns and their potential to drive effector function via Fcγ receptors and complement pathways. We identify unique and dynamic pro-inflammatory Fc compositions that are distinct from those in patients with COVID-19 and convalescents. Vaccine-induced anti-Spike IgG is characterized by distinct Fab- and Fc-mediated functions between different age groups and in comparison to antibodies generated during natural viral infection. These data highlight the heterogeneity of Fc responses to SARS-CoV-2 infection and vaccination and suggest that they support long-lasting protection differently.

An Albumin-Binding Domain Peptide Confers Enhanced Immunoprotection Against Viral Myocarditis by CVB3 VP1 Vaccine.

Coxsackievirus B3 (CVB3)-induced viral myocarditis is a common clinical cardiovascular disease without effective available vaccine. In this study, we tried to potentiate the immunoprotection efficacy of our previous CVB3-specific VP1 protein vaccine by introducing a streptococcal protein G-derived, draining lymph nodes (dLNs)-targeting albumin-binding domain (ABD) peptide. We found that compared with the original VP1 vaccine, ABD-fused VP1 (ABD-VP1) vaccine gained the new ability to efficiently bind murine albumin both in vitro and in vivo, possessed a much longer serum half-life in serum and exhibited more abundance in the dLNs after immunization. Accordingly, ABD-VP1 immunization not only significantly facilitated the enrichment and maturation of dendritic cells (DCs), induced higher percentages of IFN-γ+ CD8 + cells in the dLNs, but also robustly promoted VP1-induced T cell proliferation and cytotoxic T lymphocyte (CTL) responses in the spleens. More importantly, ABD-VP1 also elicited higher percentages of protective CD44hi CD62Lhi memory T cells in dLNs and spleens. Consequently, obvious protective effect against viral myocarditis was conferred by ABD-VP1 vaccine compared to the VP1 vaccine, reflected by the less body weight loss, improved cardiac function, alleviated cardiac histomorphological changes and an increased 28-day survival rate. Our results indicated that the ABD might be a promising immune-enhancing regime for vaccine design and development.

Targeted Delivery of mRNA with One-Component Ionizable Amphiphilic Janus Dendrimers.

Targeted and efficient delivery of nucleic acids with viral and synthetic vectors is the key step of genetic nanomedicine. The four-component lipid nanoparticle synthetic delivery systems consisting of ionizable lipids, phospholipids, cholesterol, and a PEG-conjugated lipid, assembled by microfluidic or T-tube technology, have been extraordinarily successful for delivery of mRNA to provide Covid-19 vaccines. Recently, we reported a one-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) synthetic delivery system for mRNA relying on amphiphilic Janus dendrimers and glycodendrimers developed in our laboratory. Amphiphilic Janus dendrimers consist of functional hydrophilic dendrons conjugated to hydrophobic dendrons. Co-assembly of IAJDs with mRNA into dendrimersome nanoparticles (DNPs) occurs by simple injection in acetate buffer, rather than by microfluidic devices, and provides a very efficient system for delivery of mRNA to lung. Here we report the replacement of most of the hydrophilic fragment of the dendron from IAJDs, maintaining only its ionizable amine, while changing its interconnecting group to the hydrophobic dendron from amide to ester. The resulting IAJDs demonstrated that protonated ionizable amines play dual roles of hydrophilic fragment and binding ligand for mRNA, changing delivery from lung to spleen and/or liver. Replacing the interconnecting ester with the amide switched the delivery back to lung. Delivery predominantly to liver is favored by pairs of odd and even alkyl groups in the hydrophobic dendron. This simple structural change transformed the targeted delivery of mRNA mediated with IAJDs, from lung to liver and spleen, and expands the utility of DNPs from therapeutics to vaccines.

A novel recombinant protein vaccine containing the different E7 proteins of the HPV16, 18, 6, 11 E7 linked to the HIV-1 Tat (47-57) improve cytotoxic immune responses.

OBJECTIVES: Human papillomavirus infection (HPV) is the most common viral infection which is causes of cervical, penal, vulvar, anal and, oropharyngeal cancer. E7 protein of HPV is a suitable target for induction of T cell responses and controlling HPV-related cancer. The aim of the current study was to designed and evaluated a novel fusion protein containing the different E7 proteins of the HPV 16, 18, 6 and 11, linked to the cell-penetrating peptide HIV-1 Tat 49-57, in order to improve cytotoxic immune responses in in-vitro and in-vivo. RESULTS: In this study whole sequence of HPV16,18,6,11 E7-Tat (47-57) and HPV16,18,6,11 E7 cloned into the vector and expressed in E. coli (BL21). The purified protein was confirmed by SDS page and western blotting and then injected into the C57BL/6 mice. The efficiency of the fusion protein vaccine was assessed by antibody response assay, cytokine assay (IL-4 and IFN-γ), CD + 8 cytotoxicity assay and tumor challenge experiment. Result showed that fusion proteins containing Adjuvant (IFA,CFA) could express higher titer of antibody. Also, we showed that vaccination with E7-Tat and, E7-Tat-ADJ induced high frequencies of E7-specific CD8 + T cells and CD107a expression as well as IFN-γ level and enhanced long-term survival in the therapeutic animal models. CONCLUSION: Our finding suggested that this novel fusion protein vaccine was able to induce therapeutic efficacy and immunogenicity by improving CD8 + T cell in TC-1 tumor bearing mice; so this vaccine may be appreciated for research against HPV and tumor immunotherapies.

Potential 3-chymotrypsin-like cysteine protease cleavage sites in the coronavirus polyproteins pp1a and pp1ab and their possible relevance to COVID-19 vaccine and drug development.

Coronavirus (CoV) 3-chymotrypsin (C)-like cysteine protease (3CLpro ) is a target for anti-CoV drug development and drug repurposing because along with papain-like protease, it cleaves CoV-encoded polyproteins (pp1a and pp1ab) into nonstructural proteins (nsps) for viral replication. However, the cleavage sites of 3CLpro and their relevant nsps remain unclear, which is the subject of this perspective. Here, we address the subject from three standpoints. First, we explore the inconsistency in the cleavage sites and relevant nsps across CoVs, and investigate the function of nsp11. Second, we consider the nsp16 mRNA overlapping of the spike protein mRNA, and analyze the effect of this overlapping on mRNA vaccines. Finally, we study nsp12, whose existence depends on ribosomal frameshifting, and investigate whether 3CLpro requires a large number of inhibitors to achieve full inhibition. This perspective helps us to clarify viral replication and is useful for developing anti-CoV drugs with 3CLpro as a target in the current coronavirus disease 2019 (COVID-19) pandemic.

Combined use of lactic-acid-producing bacteria as probiotics and rotavirus vaccine candidates expressing virus-specific proteins.

Due to the lower efficacy of currently approved live attenuated rotavirus (RV) vaccines in developing countries, a new approach to the development of safe mucosally administered live bacterial vectors is being considered, using probiotic bacteria as an efficient delivery platform for heterologous RV antigens. Lactic acid bacteria (LAB), which are considered food-grade bacteria and normal microbiota, have been utilized throughout history as probiotics and developed since the 1990s as a delivery system for recombinant heterologous proteins. Over the last decade, LAB have frequently been used as a platform for the delivery of various RV antigens to the mucosa. Given the appropriate safety profile for neonates and providing the benefits of probiotics, recombinant LAB-based vaccines could potentially address the need for a subunit RV vaccine. The present review focuses mainly on different recombinant LAB vaccine constructs for RV and their potential as an alternative recombinant vaccine against RV disease.

Recombinant vaccines for COVID-19.

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation. Considering the crucial role of SARS-CoV-2 spike (S) glycoprotein in virus attachment, entry, and induction of neutralizing antibodies, S protein is being widely used as a target for vaccine development. Based on advances in techniques for vaccine design, inactivated, live-vectored, nucleic acid, and recombinant COVID-19 vaccines are being developed and tested for their efficacy. Phase3 clinical trials are underway or will soon begin for several of these vaccines. Assuming that clinical efficacy is shown for one or more vaccines, safety is a major aspect to be considered before deploying such vaccines to the public. The current review focuses on the recent advances in recombinant COVID-19 vaccine research and development and associated issues.

Sulfonate-isosteric replacement examined within heroin-hapten vaccine design.

Heroin overdose and addiction remain significant health and economic burdens in the world today costing billions of dollars annually. Moreover, only limited pharmacotherapeutic options are available for treatment of heroin addiction. In our efforts to combat the public health threat posed by heroin addiction, we have developed vaccines against heroin. To expand upon our existing heroin-vaccine arsenal, we synthesized new aryl and alkyl sulfonate ester haptens; namely aryl-mono-sulfonate (HMsAc) and Aryl/alkyl-di-sulfonate (H(Ds)2) as carboxyl-isosteres of heroin then compared them to our model heroin-hapten (HAc) through vaccination studies. Heroin haptens were conjugated to the carrier protein CRM197 and the resulting CRM-immunoconjugates were used to vaccinate Swiss Webster mice following an established immunization protocol. Binding studies revealed that the highest affinity anti-heroin antibodies were generated by the HMsAc vaccine followed by the HAc and H(Ds)2 vaccines, respectively (HMsAc > HAc≫HDs2). However, neither the HMsAc nor H(Ds)2 vaccines were able to generate high affinity antibodies to the psychoactive metabolite 6-acetyl morphine (6-AM), in comparison to the HAc vaccine. Blood brain bio-distribution studies supported these binding results with vaccine efficiency following the trend HAc > HMsAc â‰« H(Ds)2 The work described herein provides insight into the use of hapten-isosteric replacement in vaccine drug design.

Immunization of mice against alpha, beta, and epsilon toxins of Clostridium perfringens using recombinant rCpa-b-x expressed by Bacillus subtilis.

The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens.

A Heterologous Viral Protein Scaffold for Chimeric Antigen Design: An Example PCV2 Virus Vaccine Candidate.

Recombinant vaccines have low-cost manufacturing, regulatory requirements, and reduced side effects compared to attenuated or inactivated vaccines. In the porcine industry, post-weaning multisystemic disease syndrome generates economic losses, characterized by progressive weight loss and weakness in piglets, and it is caused by porcine circovirus type 2 (PCV2). We designed a chimeric antigen (Qm1) to assemble the main exposed epitopes of the Cap-PCV2 protein on the capsid protein of the tobacco necrosis virus (TNV). This design was based on the Cap-N-terminal of an isolated PCV2 virus obtained in Chile. The virus was characterized, and the sequence was clustered within the PCV2 genotype b clade. This chimeric protein was expressed as inclusion bodies in both monomeric and multimeric forms, suggesting a high-molecular-weight aggregate formation. Pigs immunized with Qm1 elicited a strong and specific antibody response, which reduced the viral loads after the PCV2 challenge. In conclusion, the implemented design allowed for the generation of an effective vaccine candidate. Our proposal could be used to express the domains or fragments of antigenic proteins, whose structural complexity does not allow for low-cost production in Escherichia coli. Hence, other antigen domains could be integrated into the TNV backbone for suitable antigenicity and immunogenicity. This work represents new biotechnological strategies, with a reduction in the costs associated with vaccine development.

Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis.

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.

Adenoviral Vectors as Vaccines for Emerging Avian Influenza Viruses.

It is evident that the emergence of infectious diseases, which have the potential for spillover from animal reservoirs, pose an ongoing threat to global health. Zoonotic transmission events have increased in frequency in recent decades due to changes in human behavior, including increased international travel, the wildlife trade, deforestation, and the intensification of farming practices to meet demand for meat consumption. Influenza A viruses (IAV) possess a number of features which make them a pandemic threat and a major concern for human health. Their segmented genome and error-prone process of replication can lead to the emergence of novel reassortant viruses, for which the human population are immunologically naïve. In addition, the ability for IAVs to infect aquatic birds and domestic animals, as well as humans, increases the likelihood for reassortment and the subsequent emergence of novel viruses. Sporadic spillover events in the past few decades have resulted in human infections with highly pathogenic avian influenza (HPAI) viruses, with high mortality. The application of conventional vaccine platforms used for the prevention of seasonal influenza viruses, such as inactivated influenza vaccines (IIVs) or live-attenuated influenza vaccines (LAIVs), in the development of vaccines for HPAI viruses is fraught with challenges. These issues are associated with manufacturing under enhanced biosafety containment, and difficulties in propagating HPAI viruses in embryonated eggs, due to their propensity for lethality in eggs. Overcoming manufacturing hurdles through the use of safer backbones, such as low pathogenicity avian influenza viruses (LPAI), can also be a challenge if incompatible with master strain viruses. Non-replicating adenoviral (Ad) vectors offer a number of advantages for the development of vaccines against HPAI viruses. Their genome is stable and permits the insertion of HPAI virus antigens (Ag), which are expressed in vivo following vaccination. Therefore, their manufacture does not require enhanced biosafety facilities or procedures and is egg-independent. Importantly, Ad vaccines have an exemplary safety and immunogenicity profile in numerous human clinical trials, and can be thermostabilized for stockpiling and pandemic preparedness. This review will discuss the status of Ad-based vaccines designed to protect against avian influenza viruses with pandemic potential.

Establishment of an induced pluripotent cell line from Taiwan black silkie chick embryonic fibroblasts for replication-incompetent virus production.

The objective of this study was to establish a versatile cell line for replication-incompetent virus production and inactivation with formaldehyde to generate a model of cell-based vaccine manufacturing process. To achieve this goal, we took advantage of the easily accessed chick embryonic fibroblasts. Nine-day old chick embryonic fibroblasts were obtained and subjected to be transduced with a set of lentivirus to develop a chick induced pluripotent stem (ciPS) cell line. Morphological features, positive periodic acid-Schiff staining as well as strong immunocytofluorescence of alkaline phosphatase, intestinal (ALPI) and POU class 5 homeobox 1 (POU5F1) proteins suggested that these chick embryonic fibroblasts have been transformed into ciPS cells. Further differentiation and immunocytofluorescence assays confirmed that this ciPS cell line possesses capacities and potentials to form embryoid bodies, differentiate into all three embryonic layers: ectoderm, mesoderm and endoderm with evidence of strongly positive and specific molecular markers. Immunoblot analysis next demonstrated that through recombinant DNA technology and the 2nd generation lentiviral transfer system, the goose hemagglutinin gene (H5) gene was packaged into the replication-incompetent virus and highly expressed in a bladder cancer-derived cell line, T24, after transduction. The titer of ciPS-generated replication-incompetent virus is comparable to that from the Phoenix-AMPHO cell line, which is a commercial and high productive retrovirus producer. Our study successfully established a ciPS cell line which is able to produce replication-incompetent virus, providing a new strategy for cell-based vaccine production after virus inactivation.

Pichia pastoris-expressed Zika virus envelope domain III on a virus-like particle platform: design, production and immunological evaluation.

Zika virus (ZIKV) is an arbovirus which shares antigenic similarity and the mosquito vector with dengue viruses (DENVs). ZIKV is a neurotropic virus capable of causing congenital neurodevelopmental birth defects. As ZIKV antibodies (Abs) can potentially enhance infection by DENVs, a preventive ZIKV vaccine must be designed to eliminate antibody dependent enhancement of infection. We developed a Zika Subunit Vaccine (ZSV) consisting of two proteins, ZS and S, in a genetically pre-determined ratio of 1:4, using the methylotrophic yeast Pichia pastoris. ZS is an in-frame fusion of ZIKV envelope domain III with the Hepatitis B virus (HBV) surface antigen, and S is the un-fused HBV surface antigen. Using specific monoclonal Abs we showed the presence of ZS and S in the co-purified material which were found to co-assemble into virus-like particles (VLPs), based on dynamic light scattering and electron microscopic analyses. These VLPs were immunogenic in BALB/c mice, eliciting Abs capable of neutralizing ZIKV reporter virus particles. Further, the VLP-induced Abs did not enhance a sub-lethal DENV-2 challenge in AG129 mice. This important safety feature, coupled to the well-documented advantage of P. pastoris expression system, warrants further exploration of ZSV VLP as a possible vaccine candidate.

M918: A Novel Cell Penetrating Peptide for Effective Delivery of HIV-1 Nef and Hsp20-Nef Proteins into Eukaryotic Cell Lines.

BACKGROUND: HIV-1 Nef protein is a possible attractive target in the development of therapeutic HIV vaccines including protein-based vaccines. The most important disadvantage of protein-based vaccines is their low immunogenicity which can be improved by heat shock proteins (Hsps) as an immunomodulator, and cell-penetrating peptides (CPPs) as a carrier. METHODS: In this study, the HIV-1 Nef and Hsp20-Nef proteins were generated in E.coli expression system for delivery into the HEK-293T mammalian cell line using a novel cell-penetrating peptide, M918, in a non-covalent fashion. The size, zeta potential and morphology of the peptide/protein complexes were studied by scanning electron microscopy (SEM) and Zeta sizer. The efficiency of Nef and Hsp20-Nef transfection using M918 was evaluated by western blotting in HEK-293T cell line. RESULTS: The SEM data confirmed the formation of discrete nanoparticles with a diameter of approximately 200-250 nm and 50-80 nm for M918/Nef and M918/Hsp20-Nef, respectively. The dominant band of ~ 27 kDa and ~ 47 kDa was detected in the transfected cells with the Nef/ M918 and Hsp20-Nef/ M918 nanoparticles at a molar ratio of 1:20 using anti-HIV-1 Nef monoclonal antibody. These bands were not detected in the un-transfected and transfected cells with Nef or Hsp20- Nef protein alone indicating that M918 could increase the penetration of Nef and Hsp20-Nef proteins into the cells. CONCLUSION: These data suggest that M918 CPP can be used to enter HIV-1 Nef and Hsp20-Nef proteins inside mammalian cells efficiently as a promising approach in HIV-1 vaccine development.

Scaffold-mediated delivery for non-viral mRNA vaccines.

mRNA is increasingly being recognized as a promising alternative to pDNA in gene vaccinations. Only recently, owing to the needs of cancer immunotherapies, has the biomaterials/gene delivery community begun to develop new biomaterial strategies for immunomodulation. Here, we report a novel way to use implantable porous scaffolds as a local gene delivery depot to enhance mRNA vaccine immunization in vitro, and in vivo when compared with conventional bolus injections. We first evaluated transfection efficiencies of single-stranded mRNA condensed and charge neutralized with two lipids (Lipofectamine Messenger MAXTM LM-MM and StemfectTM SF) and two cationic polymers (in vivo-jetPEI™, Poly (ß-amino ester)) as gene carriers. As SF demonstrated highest in vitro transfection and cell viability, it was selected for subsequent porous polymer scaffold-loading trials. Enhanced in vitro transfection of SF:mRNA nanoparticle-loaded poly (2-hydroxyethyl methacrylate) (pHEMA) scaffolds was also observed with a DC2.4 cell line. Improved sustained local release and local transgene expression were also demonstrated with SF:mRNA nanoparticle-loaded pHEMA scaffolds in vivo compared with bolus injections. Our results suggest that mRNA polyplex-loaded scaffolds may be a superior alternative to either repeated bolus immunizations or ex vivo transfection cell immunotherapies.