Lipid nanoparticle-encapsulated DNA vaccine robustly induce superior immune responses to the mRNA vaccine in Syrian hamsters.

DNA vaccines for infectious diseases and cancer have been explored for years. To date, only one DNA vaccine (ZyCoV-D) has been authorized for emergency use in India. DNA vaccines are inexpensive and long-term thermostable, however, limited by the low efficiency of intracellular delivery. The recent success of mRNA/lipid nanoparticle (LNP) technology in the coronavirus disease 2019 (COVID-19) pandemic has opened a new application for nucleic acid-based vaccines. Here, we report that plasmid encoding a trimeric spike protein with LNP delivery (pTS/LNP), similar to those in Moderna's COVID-19 vaccine, induced more effective humoral responses than naked pTS or pTS delivered via electroporation. Compared with TSmRNA/LNP, pTS/LNP immunization induced a comparable level of neutralizing antibody titers and significant T helper 1-biased immunity in mice; it also prolonged the maintenance of higher antigen-specific IgG and neutralizing antibody titers in hamsters. Importantly, pTS/LNP immunization exhibits enhanced cross-neutralizing activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and protects hamsters from the challenge of SARS-CoV-2 (Wuhan strain and the Omicron BA.1 variant). This study indicates that pDNA/LNPs as a promising platform could be a next-generation vaccine technology.

A broad-spectrum pneumococcal vaccine induces mucosal immunity and protects against lethal Streptococcus pneumoniae challenge.

Pneumococcal disease is a major threat to public health globally, impacting individuals across all age groups, particularly infants and elderly individuals. The use of current vaccines has led to unintended consequences, including serotype replacement, leading to a need for a new approach to combat pneumococcal disease. A promising solution is the development of a broad-spectrum pneumococcal vaccine. In this study, we present the development of a broad-spectrum protein-based pneumococcal vaccine that contains three pneumococcal virulence factors: rlipo-PsaA (lipidated form), rPspAΔC (truncated form), and rPspCΔC (truncated form). Intranasal immunization with rlipo-PsaA, rPspAΔC, and rPspCΔC (LAAC) resulted in significantly higher IgG titres than those induced by administration of nonlipidated rPsaA, rPspAΔC, and rPspCΔC (AAC). Furthermore, LAAC immunization induced the production of higher IgA titres in vaginal washes, feces, and sera in mice, indicating that LAAC can induce systemic mucosal immunity. In addition, administration of LAAC also induced Th1/Th17-biased immune responses and promoted opsonic phagocytosis of Streptococcus pneumoniae strains of various serotypes, implying that the immunogenicity of LAAC immunization provides a protective effect against pneumococcal infection. Importantly, challenge data showed that the LAAC-immunized mice had a reduced bacterial load not only for several serotypes of the 13-valent conjugate pneumococcal vaccine (PCV13) but also for selected non-PCV13 serotypes. Consistently, LAAC immunization increased the survival rate of mice after bacterial challenge with both PCV13 and non-PCV13 serotypes. In conclusion, our protein-based pneumococcal vaccine provides protective effects against a broad spectrum of Streptococcus pneumoniae serotypes.

Co-delivery of a trimeric spike DNA and protein vaccine with aluminum hydroxide enhanced Th1-dominant humoral and cellular immunity against SARS-CoV-2.

Protein subunit vaccines have been used as prophylactic vaccines for a long time. The well-established properties of these vaccines make them the first choice for the coronavirus disease 2019 (COVID-19) outbreak. However, it is not easy to develop a protein vaccine that induces cytotoxic T lymphocyte responses and requires a longer time for manufacturing, which limits the usage of this vaccine type. Here, we report the combination of a recombinant spike (S)-trimer protein with a DNA vaccine-encoded S protein as a novel COVID-19 vaccine. The recombinant S protein was formulated with different adjuvants and mixed with the DNA plasmid before injection. We found that the recombinant S protein formulated with the adjuvant aluminum hydroxide and mixed with the DNA plasmid could enhance antigen-specific antibody titers, neutralizing antibody titers. We further evaluated the IgG2a/IgG1 isotype and cytokine profiles of the specific boosted T-cell response, which indicated that the combined vaccine induced a T-helper 1 cell-biased immune response. Immunized hamsters were challenged with severe acute respiratory syndrome coronavirus 2, and the body weight of the hamsters that received the recombinant S protein with aluminum hydroxide and/or the DNA plasmid was not reduced. Alternatively, those that received control or only the DNA plasmid immunization were reduced. Interestingly, after the third day of the viral load in the lungs, the viral challenge could not be detected in hamsters immunized with the recombinant S protein in aluminum hydroxide mixed with DNA (tissue culture infectious dose < 10). The viral load in the lungs was 109 , 106 , and 107 for the phosphate-buffered saline, protein in aluminum hydroxide, and DNA-only immunizations, respectively. These results indicated that antiviral mechanisms neutralizing antibodies play important roles. Furthermore, we found that the combination of protein and DNA vaccination could induce relatively strong CD8+ T-cell responses. In summary, the protein subunit vaccine combined with a DNA vaccine could induce strong CD8+ T-cell responses to increase antiviral immunity for disease control.

Recombinant lipidated FLIPr effectively enhances mucosal and systemic immune responses for various vaccine types.

Formyl peptide receptor-like 1 inhibitor protein (FLIPr) is an immune evasion protein produced by Staphylococcus aureus, and FLIPr is a potential vaccine candidate for reducing Staphylococcus aureus virulence and biofilm formation. We produced recombinant lipidated FLIPr (rLF) to increase the immunogenicity of FLIPr and showed that rLF alone elicited potent anti-FLIPr antibody responses to overcome the FLIPr-mediated inhibition of phagocytosis. In addition, rLF has potent immunostimulatory properties. We demonstrated that rLF is an effective adjuvant. When an antigen is formulated with rLF, it can induce long-lasting antigen-specific immune responses and enhance mucosal and systemic antibody responses as well as broad-spectrum T-cell responses in mice. These findings support further exploration of rLF in the clinic as an adjuvant for various vaccine types with extra benefits to abolish FLIPr-mediated immunosuppressive effects.

Omicron-specific mRNA vaccine induced cross-protective immunity against ancestral SARS-CoV-2 infection with low neutralizing antibodies.

The major challenge in COVID-19 vaccine effectiveness is immune escape by SARS-CoV-2 variants. To overcome this, an Omicron-specific messenger RNA (mRNA) vaccine was designed. The extracellular domain of the spike of the Omicron variant was fused with a modified GCN4 trimerization domain with low immunogenicity (TSomi). After immunization with TSomi mRNA in hamsters, animals were challenged with SARS-CoV-2 virus. The raised nonneutralizing antibodies or cytokine secretion responses can recognize both Wuhan S and Omicron S. However, the raised antibodies neutralized SARS-CoV-2 Omicron virus infection but failed to generate Wuhan virus neutralizing antibodies. Surprisingly, TSomi mRNA immunization protected animals from Wuhan virus challenge. These data indicated that non-neutralizing antibodies or cellular immunity may play a more important role in vaccine-induced protection than previously believed. Next-generation COVID-19 vaccines using the Omicron S antigen may provide sufficient protection against ancestral or current SARS-CoV-2 variants.

A DNA vaccine candidate delivered by an electroacupuncture machine provides protective immunity against SARS-CoV-2 infection.

A major challenge in the use of DNA vaccines is efficient DNA delivery in vivo. Establishing a safe and efficient electric transfer method is the key to developing rapid DNA vaccines against emerging infectious diseases. To overcome the complexity of designing new electric transfer machines for DNA delivery, a clinically approved electric transfer machine could be considered as an alternative. Here, we report an electroacupuncture machine-based method for DNA vaccine delivery after intramuscular injection of the COVID-19 DNA vaccine. The S gene of SARS-CoV-2 in the pVAX1 plasmid (pSARS2-S) was used as an antigen in this study. We optimized the clinically used electroacupuncture machine settings for efficient induction of the neutralizing antibody titer after intramuscular injection of pSARS2-S in mice. We found that pSARS2-S immunization at 40 Vpp for 3-5 s could induce high neutralizing antibody titers and Th1-biased immune responses. IFN-γ/TNF-α-secreting CD4+ and CD8+ T cells were also observed in the DNA vaccination group but not in the recombinant protein vaccination group. T-cell epitope mapping shows that the major reactive epitopes were located in the N-terminal domain (a.a. 261-285) and receptor-binding domain (a.a. 352-363). Importantly, pSARS2-S immunization in hamsters could induce protective immunity against SARS-CoV-2 challenge in vivo. In the preclinical toxicology study, blood biochemistry, hematology, and DNA persistence analysis reveal that the DNA delivery method is safe. Furthermore, the raised antisera could also cross-neutralize different variants of concern. These findings suggest that DNA vaccination using an electroacupuncture machine is feasible for use in humans in the future.

A Polypeptide of Tumor-Associated Antigen L6 with Intrinsic Adjuvant Activity Enhances Antitumor Immunity.

Peptide vaccines are safe, and aim to elicit and expand tumor-specific immunity so as to eradicate tumors. However, achieving strong and long-lasting anti-tumor immunity with peptide vaccines for the antigen-specific treatment of cancer is challenging, in part because their efficacy depends on strong adjuvants or immunomodulators. We approached this problem by conjugating an epitope-based cancer vaccine with a lipidated sequence (an immunomodulator) to elicit a strong immune response. Lipidated and non-lipidated polyepitope proteins were generated that contained the universal T helper cell epitope (pan-DR), B cell epitopes, and the extended loop sequence of extracellular domain 2 of tumor-associated antigen L6 (TAL6). We show that the lipidated polyepitope cancer vaccine can activate bone marrow-derived dendritic cells, and trigger effective antigen-specific antibody and T helper cell responses, more effectively than the non-lipidated vaccine. Moreover, potent T cell immune responses were elicited in mice inoculated with the lipidated polyepitope cancer vaccine, providing protective antitumor immunity in mice bearing TAL6 tumors. Our study demonstrates that a lipidated polyepitope cancer vaccine could be employed to generate potent anti-tumor immune responses, including humoral and cellular immunity, which could be beneficial in the treatment of TAL6+ cancer.

Domain 4 of pneumolysin from Streptococcus pneumoniae is a multifunctional domain contributing TLR4 activating and hemolytic activity.

The pneumolysin (Ply) protein of Streptococcus pneumoniae is composed of four domains and possesses several different but related activities. In this study, recombinant Ply and two truncated forms, Ply domain 1-3 and Ply domain 4 (rPly4), were expressed and characterized regarding their participation in apoptosis, the stimulation of cytokine production, hemolytic activity and virulence. rPly4 activated murine bone marrow-derived dendritic cells in a Toll-like receptor (TLR) 4-dependent manner. The rPly4 alone was able to produce hemolytic activity at high concertation and penetrate the lipid bilayer. We further demonstrated that domain 4 of Ply involved in the virulence of the bacteria in mouse model. In the absence of apoptotic activity, the virulence level caused by rPly4 was similar to that of full length Ply. Our data suggested that domain 4 of Ply alone with TLR4 agonist and hemolytic activity may play roles in virulence of Streptococcus pneumoniae.

A therapeutic vaccine targeting HPV E6/E7 with intrinsic Toll-like receptor 2 agonist activity induces antitumor immunity.

The E6 and E7 oncoproteins of human papillomavirus (HPV) are ideal targets for developing immunotherapeutic approaches to treat HPV-associated tumors. Our previous studies showed that a recombinant lipidated HPV16 E7 mutant (rlipo-E7m) with inactivation of the E7 oncogenic functions can activate antigen presenting cells through Toll-like receptor 2 (TLR2) and induce antitumor immunity. Given that some HPV-associated tumors overexpress E6 but not E7, it is necessary to include therapeutic agents containing HPV E6 in therapeutic vaccine development to broaden the utility of the vaccine. In this study, we further incorporated a mutant HPV16 E6 (E6m) into rlipo-E7m to generate rlipo-E6mE7m, which could elicit both E6- and E7-specific immune responses after immunization. The rlipo-E6mE7m immunization induced higher levels of T cell proliferation and cytotoxic T lymphocyte response than the nonlipidated recombinant E6mE7m (rE6mE7m) immunization. Accordingly, a single-dose administration of rlipo-E6mE7m at day 7 after tumor inoculation in mice showed complete inhibition of tumor growth, whereas administration of rE6mE7m did not. These results demonstrated that rlipo-E6mE7m could be used in tumors with E6 and/or E7 expression via the induction of E6- and E7-specific immunity.

Immunological study of HA1 domain of hemagglutinin of influenza H5N1 virus.

The neutralization titer of a hemagglutinin (HA)-specific neutralizing antibody against new isolates reflect both the antigenic drift and the conformation status of HA protein in these new influenza viruses. Since most antigenic sites are in the HA1 domain of HA, using HA1 domain of influenza virus as antigen is of great importance in vaccine development. In this study, we investigate different purification processes for optimizing the immunological properties of an Escherichia coli-expressed HA1 domain (rH5HA1) of influenza H5N1 virus. rH5HA1 was expressed as inclusion bodies and extracted with 6M guanidine hydrochloride (GnHCl)/PBS buffer. The best condition for generating HA1-specific neutralization determinants is on-column oxidative refolding procedures with GSH/GSSG and l-arginine buffer. Others refolding procedures such as using high-pH buffer and/or different detergent solubilizations were found to be ineffective producing neutralization epitope recognized by a HA1-specific neutralizing monoclonal antibody that was raised against H5N1 virus.

A novel technology for the production of a heterologous lipoprotein immunogen in high yield has implications for the field of vaccine design.

We have developed a novel platform technology that can express high levels of recombinant lipoproteins with intrinsic adjuvant properties. In this study, Ag473 (a lipoprotein from Neisseria meningitidis) can be produced in high yields using Escherichia coli strain C43 (DE3). After testing a non-lipoimmunogen (E3, from dengue virus) fused with different lipid signal peptides from other lipoproteins as well as Ag473 fragments of different lengths, we identified that the fusion sequence has to contain at least the N-terminal 40 residues, D1, of Ag473 to achieve high expression levels of the recombinant lipo-immunogen (rlipo-D1E3). The rlipo-D1E3 was found to elicit stronger anti-E3 and virus neutralizing antibody responses in animal studies than those from rE3 alone or rE3 formulated with alum adjuvant. These results have successfully demonstrated the merit of lipo-immunogens for novel vaccine development.