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.

Assessment of adjuvantation strategy of lipid squalene nanoparticles for enhancing the immunogenicity of a SARS-CoV-2 spike subunit protein against COVID-19.

Vaccination is regarded as the most effective intervention for controlling the coronavirus disease 2019 (COVID-19) pandemic. The objective of this study is to provide comprehensive information on lipid squalene nanoparticle (SQ@NP)-adjuvanted COVID-19 vaccines regarding modulating immune response and enhancing vaccine efficacy. After being adjuvanted with SQ@NP, the SARS-CoV-2 spike (S) subunit protein was intramuscularly (i.m.) administered to mice. Serum samples investigated by ELISA and virus neutralizing assay showed that a single-dose SQ@NP-adjuvanted S-protein vaccine can induce antigen-specific IgG and protective antibodies comparable with those induced by two doses of nonadjuvanted protein vaccine. When the mice received a boosting vaccine injection, anamnestic response was observed in the groups of adjuvanted vaccine. Furthermore, the secretion of cytokines in splenocytes, such as interferon (IFN)-γ, interleukin (IL)-5 and IL-10, was significantly enhanced after adjuvantation of S-protein vaccine with SQ@NP; however, this was not the case for the vaccine adjuvanted with conventional aluminum mineral salts. Histological examination of injection sites showed that the SQ@NP-adjuvanted vaccine was considerably well tolerated following i.m. injection in mice. These results pave the way for the performance tuning of optimal vaccine formulations against COVID-19.

Depletion of tumor-associated macrophages enhances the anti-tumor immunity induced by a Toll-like receptor agonist-conjugated peptide.

It has been reported that lipopeptides can be used to elicit cytotoxic T lymphocyte (CTL) responses against viral diseases and cancer. In our previous study, we determined that mono-palmitoylated peptides can enhance anti-tumor responses in the absence of adjuvant activity. To investigate whether di-palmitoylated peptides with TLR2 agonist activity are able to induce anti-tumor immunity, we synthesized a di-palmitic acid-conjugated long peptide that contains a murine CTL epitope of HPV E749-57 (Pam2IDG). Pam2IDG stimulated the maturation of bone marrow-derived dendritic cells (BMDCs) through TLR2/6. After immunization, Pam2IDG induced higher levels of T cell responses than those obtained with its non-lipidated counterpart (IDG). In the prophylactic model, Pam2IDG immunization completely inhibited tumor growth, whereas IDG immunization was unable to inhibit tumor growth. However, Pam2IDG immunization could not effectively inhibit the growth of established tumors. Therefore, we further investigated whether the depletion of immunosuppressive factors could improve the therapeutic effects of Pam2IDG. Our data indicate that treatment with Pam2IDG combined with clodronate/liposome delays tumor growth and increases the survival rate. We also observed that the therapeutic effects of Pam2IDG are improved by diminishing the function of tumor-associate macrophages (TAMs) and through the use of an IL10 receptor blocking antibody or a Cyclooxygenase 2 (Cox-2) inhibitor. In conclusion, the depletion of TAMs may enhance the anti-tumor immunity of a TLR2 agonist-conjugated peptide.

A consensus envelope protein domain III can induce neutralizing antibody responses against serotype 2 of dengue virus in non-human primates.

We have previously demonstrated that vaccination with a subunit dengue vaccine containing a consensus envelope domain III with aluminum phosphate elicits neutralizing antibodies against all four serotypes of dengue virus in mice. In this study, we evaluated the immunogenicity of the subunit dengue vaccine in non-human primates. After vaccination, monkeys that received the subunit vaccine with aluminum phosphate developed a significantly strong and long-lasting antibody response. A specific T cell response with cytokine production was also induced, and this correlated with the antibody response. Additionally, neutralizing antibodies against serotype 2 were detected in two of three monkeys. The increase in serotype-2-specific antibody titers and avidity observed in these two monkeys suggested that a serotype-2-biased antibody response occurs. These data provide evidence that a protective neutralizing antibody response was successfully elicited in non-human primates by the dengue subunit vaccine with aluminum phosphate adjuvant.

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 dengue vaccine candidate that induces cross-neutralizing antibodies and memory immunity.

A novel dengue vaccine candidate comprised of a consensus dengue virus envelope protein domain III (cED III) was developed to fight against dengue virus infection. The amino acid sequence of this novel cED III was obtained by alignment of amino acid sequences from different isolates of the four serotypes of dengue viruses. A proof-of-concept study demonstrated that BALB/c mice immunized with the recombinant cED III developed neutralizing antibodies against all serotypes of dengue virus. Moreover, formulation of recombinant cED III with aluminum phosphate could induce long-lasting antibody responses and anamnestic neutralizing antibody responses following challenge with dengue virus at week 28 after priming. These results demonstrate the possibility of developing a single tetravalent vaccine against dengue viral infections.