Heterologous vaccination utilizing viral vector and protein platforms confers complete protection against SFTSV.

Severe fever with thrombocytopenia syndrome virus was first discovered in 2009 as the causative agent of severe fever with thrombocytopenia syndrome. Despite its potential threat to public health, no prophylactic vaccine is yet available. This study developed a heterologous prime-boost strategy comprising priming with recombinant replication-deficient human adenovirus type 5 (rAd5) expressing the surface glycoprotein, Gn, and boosting with Gn protein. This vaccination regimen induced balanced Th1/Th2 immune responses and resulted in potent humoral and T cell-mediated responses in mice. It elicited high neutralizing antibody titers in both mice and non-human primates. Transcriptome analysis revealed that rAd5 and Gn proteins induced adaptive and innate immune pathways, respectively. This study provides immunological and mechanistic insight into this heterologous regimen and paves the way for future strategies against emerging infectious diseases.

Induction of protective immune responses against a lethal Zika virus challenge post-vaccination with a dual serotype of recombinant vesicular stomatitis virus carrying the genetically modified Zika virus E protein gene.

The development of a vaccine to prevent Zika virus (ZIKV) infection has been one of the priorities in infectious disease research in recent years. There have been numerous attempts to develop an effective vaccine against ZIKV. It is imperative to choose the safest and the most effective ZIKV vaccine from all candidate vaccines to control this infection globally. We have employed a dual serotype of prime-boost recombinant vesicular stomatitis virus (VSV) vaccine strategy, to develop a ZIKV vaccine candidate, using a type 1 IFN-receptor knock-out (Ifnar-/-) mouse model for challenge studies. Prime vaccination with an attenuated recombinant VSV Indiana serotype (rVSVInd) carrying a genetically modified ZIKV envelope (E) protein gene followed by boost vaccination with attenuated recombinant VSV New Jersey serotype (rVSVNJ) carrying the same E gene induced robust adaptive immune responses. In particular, rVSV carrying the ZIKV E gene with the honeybee melittin signal peptide (msp) at the N terminus and VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the C terminus of the E gene induced strong protective immune responses. This vaccine regimen induced highly potent neutralizing antibodies and T cell responses in the absence of an adjuvant and protected Ifnar-/- mice from a lethal dose of the ZIKV challenge.

Cross-Protection against MERS-CoV by Prime-Boost Vaccination Using Viral Spike DNA and Protein.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness and has a high mortality of ∼34%. However, since its discovery in 2012, an effective vaccine has not been developed for it. To develop a vaccine against multiple strains of MERS-CoV, we targeted spike glycoprotein (S) using prime-boost vaccination with DNA and insect cell-expressed recombinant proteins for the receptor-binding domain (RBD), S1, S2, SΔTM, or SΔER. Our S subunits were generated using an S sequence derived from the MERS-CoV EMC/2012 strain. We examined humoral and cellular immune responses of various combinations with DNA plasmids and recombinant proteins in mice. Mouse sera immunized with SΔER DNA priming/SΔTM protein boosting showed cross-neutralization against 15 variants of S-pseudovirions and the wild-type KOR/KNIH/002 strain. In addition, these immunizations provided full protection against the KOR/KNIH/002 strain challenge in human DPP4 knock-in mice. These findings suggest that vaccination with the S subunits derived from one viral strain can provide cross-protection against variant MERS-CoV strains with mutations in S. DNA priming/protein boosting increased gamma interferon production, while protein-alone immunization did not. The RBD subunit alone was insufficient to induce neutralizing antibodies, suggesting the importance of structural conformation. In conclusion, heterologous DNA priming with protein boosting is an effective way to induce both neutralizing antibodies and cell-mediated immune responses for MERS-CoV vaccine development. This study suggests a strategy for selecting a suitable platform for developing vaccines against MERS-CoV or other emerging coronaviruses.IMPORTANCE Coronavirus is an RNA virus with a higher mutation rate than DNA viruses. Therefore, a mutation in S-protein, which mediates viral infection by binding to a human cellular receptor, is expected to cause difficulties in vaccine development. Given that DNA-protein vaccines promote stronger cell-mediated immune responses than protein-only vaccination, we immunized mice with various combinations of DNA priming and protein boosting using the S-subunit sequences of the MERS-CoV EMC/2012 strain. We demonstrated a cross-protective effect against wild-type KOR/KNIH/002, a strain with two mutations in the S amino acids, including one in its RBD. The vaccine also provided cross-neutralization against 15 different S-pseudotyped viruses. These suggested that a vaccine targeting one variant of S can provide cross-protection against multiple viral strains with mutations in S. The regimen of DNA priming/Protein boosting can be applied to the development of other coronavirus vaccines.

Characterization of novel monoclonal antibodies against MERS-coronavirus spike protein.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes severe pulmonary infection, with ∼35 % mortality. Spike glycoprotein (S) of MERS-CoV is a key target for vaccines and therapeutics because S mediates viral entry and membrane-fusion to host cells. Here, four different S subunit proteins, receptor-binding domain (RBD; 358-606 aa), S1 (1-751 aa), S2 (752-1296 aa), and SΔTM (1-1296 aa), were generated using the baculoviral system and immunized in mice to develop neutralizing antibodies. We developed 77 hybridomas and selected five neutralizing mAbs by immunization with SΔTM against MERS-CoV EMC/2012 strain S-pseudotyped lentivirus. However, all five monoclonal antibodies (mAb) did not neutralize the pseudotyped V534A mutation. Additionally, one mAb RBD-14F8 did not show neutralizing activity against pseudoviruses with amino acid substitution of L506 F or D509 G (England1 strain, EMC/2012 L506 F, and EMC/2012 D509 G), and RBD-43E4 mAb could not neutralize the pseudotyped I529 T mutation, while three other neutralizing mAbs showed broad neutralizing activity. This implies that the mutation in residue 506-509, 529, and 534 of S is critical to generate neutralization escape variants of MERS-CoV. Interestingly, all five neutralizing mAbs have binding affinity to RBD, although most mAbs generated by RBD did not have neutralizing activity. Additionally, chimeric antibodies of RBD-14F8 and RBD-43E4 with human Fc and light chain showed neutralizing effect against wild type MERS-CoV KOR/KNIH/002, similar to the original mouse mAbs. Thus, our mAbs can be utilized for the identification of specific mutations of MERS-CoV.

Sublingual Immunization With an RSV G Glycoprotein Fragment Primes IL-17-Mediated Immunopathology Upon Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of serious respiratory tract disease but there is no licensed RSV vaccine. Immunopathological mechanisms have long been suspected as operating in the development of severe RSV disease and have hampered the development of safe and effective vaccines. Here, we show that unlike intranasal immunization, sublingual immunization with RSV glycoprotein fragment containing the central conserved region (Gcf) primes the host for severe disease upon RSV challenge. This increased pathology does not require replication by the challenge virus and is associated with massive infiltration of inflammatory cells, extensive cell death, and excessive mucus production in the airway and lungs. This exacerbated RSV disease primed by sublingual Gcf immunization is distinct from the immunopathology by G-expressing vaccinia virus or formalin-inactivated RSV, and preceded by prominent IL-17 production. IL-17 deficiency abolished the enhanced disease. Our results suggest a novel mechanism of RSV vaccine-induced immunopathology by IL-17, and highlights the importance of vaccination site.

Development of Safe and Non-Self-Immunogenic Mucosal Adjuvant by Recombinant Fusion of Cholera Toxin A1 Subunit with Protein Transduction Domain.

Potential use of cholera toxin (CT) as a mucosal vaccine adjuvant has been documented in a variety of animal models. However, native CT is highly toxic to be used as a mucosal adjuvant in humans. Here, we demonstrate a new approach to generate a mucosal adjuvant by replacing the B subunit of CT with HIV-1 Tat protein transduction domain (PTD), which efficiently delivers fusion proteins into the cell cytoplasm by unspecific binding to cell surface. We compared the adjuvanticity and toxicity of Tat PTD-CTA1-Tat PTD (TCTA1T) with those of CT. Our results indicate that intranasal (i.n.) delivery of ovalbumin (OVA) with TCTA1T significantly augments the OVA-specific systemic and mucosal antibody responses to levels comparable to those seen with CT adjuvant. Moreover, in vivo cytotoxic T lymphocyte activity elicited by TCTA1T was significantly higher than that elicited by a mutant TCTA1T (TmCTA1T) lacking ADP-ribosyltransferase function. In addition, coadministration of influenza M2 protein with TCTA1T conferred near complete protection against lethal influenza virus challenge. Importantly, TCTA1T, in contrast to CT, did not induce serum IgG antibody responses to itself and was shown to be nontoxic. These results suggest that TCTA1T may be a safe and effective adjuvant when given by mucosal routes.

Comparison of the adjuvanticity of two adjuvant formulations containing de-O-acylated lipooligosaccharide on Japanese encephalitis vaccine in mice.

Adjuvants are essential vaccine components used to enhance, accelerate, and/or prolong adaptive immunity against specific vaccine antigens. In this study, we compared the adjuvanticity of two adjuvant formulations containing de-O-acylated lipooligosaccharide (dLOS), a toll-like receptor 4 agonist, on the Japanese encephalitis (JE) vaccine in mice. Mice were immunized once or twice at a two-week interval with inactivated JE vaccine in the absence or presence of adjuvant. We found that both the alum- and the liposome-based formulation induced significantly faster and higher serum IgG antibody responses as compared with the non-adjuvanted vaccine after either one or two immunizations. The antibody titers of the mouse immune sera correlated with 50% plaque reduction neutralization test (PRNT50) antibody titers. In addition, the dLOS/liposome formulation was more effective in inducing a Th1-type immune response than the dLOS/alum formulation, as suggested by a strong antigen-specific interferon (IFN)-γ response. Based on these results, we suggest that both alum- and liposome-based adjuvant formulations containing dLOS may be used for the development of JE vaccines with improved immunogenicity.

Development of safe and effective RSV vaccine by modified CD4 epitope in G protein core fragment (Gcf).

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in infants and young children worldwide, but currently no safe and effective vaccine is available. The RSV G glycoprotein (RSVG), a major attachment protein, is an important target for the induction of protective immune responses during RSV infection. However, it has been thought that a CD4+ T cell epitope (a.a. 183-195) within RSVG is associated with pathogenic pulmonary eosinophilia. To develop safe and effective RSV vaccine using RSV G protein core fragment (Gcf), several Gcf variants resulting from modification to CD4+ T cell epitope were constructed. Mice were immunized with each variant Gcf, and the levels of RSV-specific serum IgG were measured. At day 4 post-challenge with RSV subtype A or B, lung viral titers and pulmonary eosinophilia were determined and changes in body weight were monitored. With wild type Gcf derived from RSV A2 (wtAGcf), although RSV A subtype-specific immune responses were induced, vaccine-enhanced disease characterized by excessive pulmonary eosinophil recruitment and body weight loss were evident, whereas wtGcf from RSV B1 (wtBGcf) induced RSV B subtype-specific immune responses without the signs of vaccine-enhanced disease. Mice immunized with Th-mGcf, a fusion protein consisting CD4+ T cell epitope from RSV F (F51-66) conjugated to mGcf that contains alanine substitutions at a.a. position 185 and 188, showed higher levels of RSV-specific IgG response than mice immunized with mGcf. Both wtAGcf and Th-mGcf provided complete protection against RSV A2 and partial protection against RSV B. Importantly, mice immunized with Th-mGcf did not develop vaccine-enhanced disease following RSV challenge. Immunization of Th-mGcf provided protection against RSV infection without the symptom of vaccine-enhanced disease. Our study provides a novel strategy to develop a safe and effective mucosal RSV vaccine by manipulating the CD4+ T cell epitope within RSV G protein.

Application of an M-cell-targeting ligand for oral vaccination induces efficient systemic and mucosal immune responses against a viral antigen.

Oral mucosal vaccination is an alternative method to overcome the pitfalls of current injection-based vaccines, such as pain and high cost of vaccination. It is a feasible and economic vaccine application, especially in developing countries. However, achieving effective antigen delivery into mucosal lymphoid organs and efficient immune stimulation are prerequisites to successful oral mucosal vaccination. One promising approach for oral mucosal vaccine development is exploring the potential of M cells via M-cell-targeting ligands that have the potential to deliver ligand-conjugated antigens into mucosal lymphoid organs and evoke conjugated-antigen-specific systemic and mucosal immune responses. Here, we investigated the M-cell-targeting ligand, Co1, in inducing specific immune responses against a pathogenic viral antigen, envelope domain III (EDIII) of dengue virus, to provide the foundation for oral mucosal vaccine development against the pathogen. After oral administration of Co1-conjugated EDIII antigens, we observed efficient antigen delivery into Peyer's patches. We also report the elicitation of EDIII-specific immunity in systemic and mucosal compartments by Co1 ligand (located in the C-terminus of EDIII). Furthermore, the antibodies induced by the ligand-conjugated EDIII antigen showed effective virus-neutralizing activity. The results of this study suggest that the M-cell-targeting strategy using Co1 ligand as a mucosal adjuvant may be applicable for developing oral vaccine candidates against pathogenic viral antigen.

M cells expressing the complement C5a receptor are efficient targets for mucosal vaccine delivery.

In the mucosal immune system, M cells are known as specialized epithelial cells that take up luminal antigens, although the receptors on M cells and the mechanism of antigen uptake into M cells are not well-understood. Here, we report the expression of the complement C5a receptor (C5aR) on the apical surface of M cells. C5ar mRNA expression in co-cultured Caco-2 human M-like cells was six-fold higher than in mono-cultured cells. C5aR expression was detected together with glycoprotein 2, an M-cell-specific protein, on the apical surface of M-like cells and mouse Peyer's patch M cells. Interestingly, after oral administration of Yersinia enterocolitica which expresses outer membrane protein H (OmpH) that is homologous to the Skp α1 domain of Escherichia coli, a ligand of C5aR, dense clustering and phosphorylation of C5aR were detected in M cells. Finally, targeted antigen delivery to M cells using C5aR as a receptor was achieved using the OmpH α1 of Y. enterocolitica such that the induction of ligand-conjugated antigen-specific immune responses was confirmed in mice after oral immunization of the OmpH ß1α1-conjugated antigen. Collectively, we identified C5aR expression on M cells and suggest that C5aR could be used as a target receptor for mucosal antigen delivery.