Potentiating the Cross-Reactive IFN-γ T Cell and Polyfunctional T Cell Responses by Heterologous GX-19N DNA Booster in Mice Primed with Either a COVID-19 mRNA Vaccine or Inactivated Vaccine.

Waning vaccine-induced immunity, coupled with the emergence of SARS-CoV-2 variants, has inspired the widespread implementation of COVID-19 booster vaccinations. Here, we evaluated the potential of the GX-19N DNA vaccine as a heterologous booster to enhance the protective immune response to SARS-CoV-2 in mice primed with either an inactivated virus particle (VP) or an mRNA vaccine. We found that in the VP-primed condition, GX-19N enhanced the response of both vaccine-specific antibodies and cross-reactive T Cells to the SARS-CoV-2 variant of concern (VOC), compared to the homologous VP vaccine prime-boost. Under the mRNA-primed condition, GX-19N induced higher vaccine-induced T Cell responses but lower antibody responses than the homologous mRNA vaccine prime-boost. Furthermore, the heterologous GX-19N boost induced higher S-specific polyfunctional CD4+ and CD8+ T cell responses than the homologous VP or mRNA prime-boost vaccinations. Our results provide new insights into booster vaccination strategies for the management of novel COVID-19 variants.

Soluble Spike DNA Vaccine Provides Long-Term Protective Immunity against SARS-CoV-2 in Mice and Nonhuman Primates.

The unprecedented and rapid spread of SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) has motivated the need for a rapidly producible and scalable vaccine. Here, we developed a synthetic soluble SARS-CoV-2 spike (S) DNA-based vaccine candidate, GX-19. In mice, immunization with GX-19 elicited not only S-specific systemic and pulmonary antibody responses but also Th1-biased T cell responses in a dose-dependent manner. GX-19-vaccinated nonhuman primates seroconverted rapidly and exhibited a detectable neutralizing antibody response as well as multifunctional CD4+ and CD8+ T cell responses. Notably, when the immunized nonhuman primates were challenged at 10 weeks after the last vaccination with GX-19, they had reduced viral loads in contrast to non-vaccinated primates as a control. These findings indicate that GX-19 vaccination provides a durable protective immune response and also support further development of GX-19 as a vaccine candidate for SARS-CoV-2.

Vaccination with single plasmid DNA encoding IL-12 and antigens of severe fever with thrombocytopenia syndrome virus elicits complete protection in IFNAR knockout mice.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease caused by SFTS virus (SFTSV) infection. Despite a gradual increase of SFTS cases and high mortality in endemic regions, no specific viral therapy nor vaccine is available. Here, we developed a single recombinant plasmid DNA encoding SFTSV genes, Gn and Gc together with NP-NS fusion antigen, as a vaccine candidate. The viral antigens were fused with Fms-like tyrosine kinase-3 ligand (Flt3L) and IL-12 gene was incorporated into the plasmid to enhance cell-mediated immunity. Vaccination with the DNA provides complete protection of IFNAR KO mice upon lethal SFTSV challenge, whereas immunization with a plasmid without IL-12 gene resulted in partial protection. Since we failed to detect antibodies against surface glycoproteins, Gn and Gc, in the immunized mice, antigen-specific cellular immunity, as confirmed by enhanced antigen-specific T cell responses, might play major role in protection. Finally, we evaluated the degree of protective immunity provided by protein immunization of the individual glycoprotein, Gn or Gc. Although both protein antigens induced a significant level of neutralizing activity against SFTSV, Gn vaccination resulted in relatively higher neutralizing activity and better protection than Gc vaccination. However, both antigens failed to provide complete protection. Given that DNA vaccines have failed to induce sufficient immunogenicity in human trials when compared to protein vaccines, optimal combinations of DNA and protein elements, proper selection of target antigens, and incorporation of efficient adjuvant, need to be further investigated for SFTSV vaccine development.

Multivalent DNA vaccine protects against genital herpes by T-cell immune induction in vaginal mucosa.

Genital herpes is one of the most common sexually transmitted infections (STIs), and it is mainly caused by the neurotropic herpes simplex virus (HSV-2). Not only does this infection cause ulcers, but HSV-2 can also stay in a latent state in the nervous system of the host throughout their lifespan. As a result, many people do not know that they harbor this infection. Moreover, HSV-2 serves as a major risk factor for human immunodeficiency virus (HIV) infection and can be transmitted to the fetus. Despite the high risk of infection and adverse effects, attempts at development of an effective vaccine for HSV-2 have not yet been successful. In this study, we developed a DNA vaccine for HSV-2 (SL-V20). This multivalent DNA vaccine effectively reduced the pathological symptoms of infection and induced efficient elimination of the virus in a mouse model. Intramuscular injection of SL-V20 led to induction of an HSV-2-specific T-cell response in the vagina, the major infection site, and in draining lymph organs. Dendritic cells (DCs), especially basic leucine zipper ATF-like transcription factor 3 (Baft3)+ DCs and partially interferon regulatory factor 4 (Irf4)+ DCs, were involved in this T-cell-mediated protective response, while B cells were dispensable for these prophylactic effects. This study demonstrates that SL-V20 offers a novel and effective vaccine against vaginal HSV-2 infection and may be applicable to patients, pending validation in clinical studies.

Utility of human papillomavirus L1 capsid protein and HPV test as prognostic markers for cervical intraepithelial neoplasia 2+ in women with persistent ASCUS /LSIL cervical cytology.

Objective: Efficient and highly predictive biomarkers reflecting the prognosis of persistent atypical squamous cells of unknown significance(ASCUS) and low grade squamous intraepithelial lesion(LSIL)s are unavailable and need to be developed urgently. We aimed to develop a predictive model for diagnosis of cervical intraepithelial neoplasia(CIN)2+ by analyzing the immunocytochemical expression of the HPV L1 capsid protein in patients with persistent ASCUS and LSIL with a high risk of HPV infection. Methods: Cervical cytology samples comprising (70 ASCUS and 215 LSIL Pap smears) were analyzed. Immunocytochemical identification of the HPV L1 capsid protein in cervical cytology samples was performed. Expression levels of HPV L1 capsid protein in cervical cytology samples were measured, and the correlation between HPV L1 expression and cervical pathologic diagnosis was evaluated. The risk for CIN2+ was calculated using the results of immunocytochemistry and the HPV DNA test. Results: Negative results for HPV L1 immunochemistry test were more frequently observed in CIN2+, and expression of the HPV L1 capsid protein was higher in CIN1 or cervicitis (Fisher's exact test, p<0.05). Diagnosis rates for CIN2+ were highest for the combination of HPV L1 capsid protein immunocytochemistry, cytology and HPV test when compared with other combinations (Akaike information criterion (AIC): 191.7, Schwarz criterion(SC): 206.3, p<0.001). Conclusion: Absence of HPV L1 capsid expression and presence of HPV type 16 or 18 infection are reliable predictors of progression to CIN2+ in patients showing persistent ASCUS and LSIL.

E2/E6 ratio and L1 immunoreactivity as biomarkers to determine HPV16-positive high-grade squamous intraepithelial lesions (CIN2 and 3) and cervical squamous cell carcinoma.

OBJECTIVE: Human papillomavirus (HPV) 16 is the most carcinogenic HPV genotype. We investigated if HPV16 L1 capsid protein and E2/E6 ratio, evaluated by cervical cytology, may be used as biomarkers of ≥cervical intraepithelial neoplasia (CIN) 2 lesions. METHODS: Cervical specimens were obtained from 226 patients with HPV16 single infection. Using cytology specimen, L1 capsid protein and E2/E6 ratio were detected and the results were compared with those of the conventional histologic analysis of cervical tissues (CIN1-3 and squamous cell carcinoma [SCC]) to evaluate the association. RESULTS: The L1 positivity of CIN2/3 was significantly lower than that of normal cervical tissue (p<0.001) and SCC demonstrated significantly lower L1 positivity than CIN1 (p<0.001). The mean E2/E6 ratios of specimens graded as SCC (0.356) and CIN2/3 (0.483) were significantly lower than those of specimens graded as CIN1 (0.786) and normal (0.793) (p<0.05). We observed that area under the receiver operating characteristic curve (AUC) for E2/E6 ratio (0.844; 95% confidence interval [CI]=0.793-0.895) was higher than that for L1 immunochemistry (0.636; 95% CI=0.562-0.711). A combination of E2/E6 ratio and L1 immunocytochemistry analyses showed the highest AUC (0.871; 95% CI=0.826-0.917) for the prediction of ≥CIN2 lesions. CONCLUSION: To our knowledge, this is the first study to validate HPV L1 capsid protein expression and decreased HPV E2/E6 ratio as valuable predictive markers of ≥CIN2 cervical lesions. Cervical cytology may be analyzed longitudinally on an outpatient basis with noninvasive procedures as against invasive conventional histologic analysis.

Intravaginal Administration of Fc-Fused IL7 Suppresses the Cervicovaginal Tumor by Recruiting HPV DNA Vaccine-Induced CD8 T Cells.

PURPOSE: The induction of tissue-localized virus-specific CD8 T-cell response is essential for the development of an effective therapeutic vaccine against genital diseases, such as cervical cancer and genital herpes. Here, we aimed to elucidate the immunologic role of IL7 in the induction of mucosal cellular immunity. EXPERIMENTAL DESIGN: IL7 was engineered through Fc fusion to enhance mucosal delivery across the genital epithelial barrier. The immunomodulatory role of IL7 was evaluated by monitoring the kinetics of various immune cells and measuring the expression of chemokines and cytokines after intravaginal administration of Fc-fused IL7 (IL7-Fc). The antitumor effects of intramuscular human papillomavirus (HPV) DNA vaccine or topical IL7-Fc alone or in a combinational regimen on mice survival were compared using a orthotopic cervical cancer model. RESULTS: Intravaginal treatment of IL7-Fc, but not native IL7, induces upregulation of chemokines (CXCL10, CCL3, CCL4, and CCL5), cytokines (IFNγ, TNFα, IL6, and IL1ß), and an adhesion molecule (VCAM-1) in the genital tract, leading to the recruitment of several leukocytes, including CD4, CD8, γδ T cells, and dendritic cells. Importantly, in this murine cervical cancer model, topical administration of IL7-Fc after intramuscular HPV DNA vaccination increases the number of HPV-specific CD8 T cells in the genital mucosa, but not in the spleen, leading to stronger antitumor activity than the HPV DNA vaccine alone. CONCLUSIONS: Our findings provide an important insight into the immunomodulatory role of IL7-Fc via topical application and the design of therapeutic vaccine regimen that induces effective genital-mucosal CD8 T-cell responses. Clin Cancer Res; 22(23); 5898-908. ©2016 AACR.

Oral immunization of haemaggulutinin H5 expressed in plant endoplasmic reticulum with adjuvant saponin protects mice against highly pathogenic avian influenza A virus infection.

Pandemics in poultry caused by the highly pathogenic avian influenza (HPAI) A virus occur too frequently globally, and there is growing concern about the HPAI A virus due to the possibility of a pandemic among humans. Thus, it is important to develop a vaccine against HPAI suitable for both humans and animals. Various approaches are underway to develop such vaccines. In particular, an edible vaccine would be a convenient way to vaccinate poultry because of the behaviour of the animals. However, an edible vaccine is still not available. In this study, we developed a strategy of effective vaccination of mice by the oral administration of transgenic Arabidopsis plants (HA-TG) expressing haemagglutinin (HA) in the endoplasmic reticulum (ER). Expression of HA in the ER resulted in its high-level accumulation, N-glycosylation, protection from proteolytic degradation and long-term stability. Oral administration of HA-TG with saponin elicited high levels of HA-specific systemic IgG and mucosal IgA responses in mice, which resulted in protection against a lethal influenza virus infection with attenuated inflammatory symptoms. Based on these results, we propose that oral administration of freeze-dried leaf powders from transgenic plants expressing HA in the ER together with saponin is an attractive strategy for vaccination against influenza A virus.

Safety and immunogenicity of therapeutic DNA vaccine with antiviral drug in chronic HBV patients and its immunogenicity in mice.

BACKGROUND & AIMS: Here, we evaluated the safety and immunogenicity of hepatitis B virus (HBV) DNA vaccine, HB-110, in mice and Korean patients with chronic hepatitis B (CHB) undergoing adefovir dipivoxil (ADV) treatment. METHODS: For animal study, mice (BALB/c or HBV transgenic) were immunized with mHB-110, and T-cell and antibody responses were evaluated. For clinical study, 27 patients randomly received either ADV alone or ADV in combination with HB-110. Liver function tests, serum HBV DNA levels and the presence of HBeAg/anti-HBe were analysed. T-cell responses were estimated by ELISPOT and FACS analysis. RESULTS: mHB-110 induced higher T-cell and antibody responses than mHB-100 in mice. No adverse effects were observed by HB-110 cotreated with ADV. HBV-specific T-cell responses were induced in a portion of patients in medium to high dose of HB-110. Interestingly, HB-110 exhibited positive effects on ALT normalization and maintenance of HBeAg seroconversion. One patient, who received high dose of HB-110 exhibited HBeAg seroconversion during vaccination, which correlated with vaccine-induced T-cell responses without ALT elevation. CONCLUSIONS: HB-110 was safe and tolerable in CHB patients. In contrast to results in animal models, HB-110 in Korean patients exhibited weaker capability of inducing HBV-specific T-cell responses and HBeAg seroconversion than HB-100 in Caucasian patients. As Asian patients, who are generally infected via vertical transmission, appeared to have higher level of immune tolerance than Caucasian, novel approaches for breaking immune tolerance rather than enhancing immunogenicity may be more urgently demanded to develop effective therapeutic HBV DNA vaccines.

Clearance of persistent HPV infection and cervical lesion by therapeutic DNA vaccine in CIN3 patients.

Here, we demonstrate that electroporation-enhanced immunization with a rationally designed HPV DNA vaccine (GX-188E), preferentially targeting HPV antigens to dendritic cells, elicits a significant E6/E7-specific IFN-γ-producing T-cell response in all nine cervical intraepithelial neoplasia 3 (CIN3) patients. Importantly, eight out of nine patients exhibit an enhanced polyfunctional HPV-specific CD8 T-cell response as shown by an increase in cytolytic activity, proliferative capacity and secretion of effector molecules. Notably, seven out of nine patients display complete regression of their lesions and viral clearance within 36 weeks of follow up. GX-188E administration does not elicit serious vaccine-associated adverse events at all administered doses. These findings indicate that the magnitude of systemic polyfunctional CD8 T-cell response is the main contributing factor for histological, cytological and virological responses, providing valuable insights into the design of therapeutic vaccines for effectively treating persistent infections and cancers in humans.

Crucial roles of interleukin-7 in the development of T follicular helper cells and in the induction of humoral immunity.

UNLABELLED: T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo, as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses. IMPORTANCE: Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.

Enhancement of antigen-specific CD8 T cell responses by co-delivery of Fc-fused CXCL11.

Chemokines have been known to play an important role in eliciting adaptive immune responses by, selectively attracting the innate cellular components to the site of antigen presentation. In this study, we demonstrated that all three CXCR3 ligands, CXCL9, CXCL10, and CXCL11, could act as a strong, genetic adjuvant. Among them, CXCL11 increased vaccine antigen-specific CD8 T cells, including, several cytokine secretions (IFN-γ and TNF-α) to a greater degree than the other two CXCR3 ligands. Fc-fusion of CXCL11 (CXCL11-Fc) induced similar but slightly higher CD8 T cell response, which, appeared to be antigen- (ovalbumin (OVA) vs. human papillomavirus 16 (HPV16) E7) and vaccine, type- (adenovirus vs. DNA vaccine) independent. In addition, the adjuvant effect of CXCL11-Fc was, further confirmed by suppressing tumor growth and extension of survival rates in a therapeutic tumor, model, which was correlated with enhanced antigen-specific CD8 T cell responses. Interestingly, the, enhanced antigen-specific CD8 T cell responses by co-delivery of CXCL11-Fc were associated with CD8, T cell proliferation, followed by increased total and effector memory T cell frequencies. Taken together, our findings provide a novel role of CXCL11 as a strong genetic adjuvant which might be used to, increase antigen-specific CD8 T cell immunity elicited by vaccination.

Complete protection against a H5N2 avian influenza virus by a DNA vaccine expressing a fusion protein of H1N1 HA and M2e.

Most influenza vaccines target hemagglutinin (HA) in order to protect the host against infection. However, theses vaccines are strain-specific due to major antigenic variations of HA. Since it is difficult to predict epidemic and pandemic strains of influenza virus, the development of effective vaccines against divergent influenza viruses is urgently needed. Although M2e-based vaccines are associated with weaker protection than HA-based vaccines that induce neutralizing antibodies against challenge virus matched-strain, the extracellular domain of Matrix 2 protein (M2e) is one of a potential broad-spectrum immunogen because it contains highly conserved sequences among influenza A viruses. In this study, M2e sequence was fused to H1N1 HA DNA (M2e-HA) and the immunogenicity and antiviral efficacy of this DNA vaccine was evaluated in response to challenge with a heterosubtypic H5N2 avian influenza virus. Compared to vaccination with HA or M2e DNA alone, vaccination with M2e-HA DNA or combination of M2e DNA and HA DNA (M2e DNA+HA DNA) induced a broad immunity without evidence of immune interference. In addition, HA-specific CD8(+) and M2e-specific T cell responses elicited by M2e-HA DNA vaccination were significantly higher than those of HA or M2e DNA vaccine alone, respectively. Following challenge with a heterosubtypic influenza virus infection, vaccination with M2e-HA DNA conferred complete protection against mortality. In combination, these results suggest that DNA vaccines expressing a fusion protein, M2e-HA, may provide an attractive approach for the development of broad-spectrum influenza vaccines.