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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and potentially fatal virus. So far, most comprehensive analyses encompassing clinical and transcriptional manifestation have concentrated on the lungs. Here, we confirmed evident signs of viral infection in the lungs and spleen of SARS-CoV-2-infected K18-hACE2 mice, which replicate the phenotype and infection symptoms in hospitalized humans. Seven days post viral detection in organs, infected mice showed decreased vital signs, leading to death. Bronchopneumonia due to infiltration of leukocytes in the lungs and reduction in the spleen lymphocyte region were observed. Transcriptome profiling implicated the meticulous regulation of distress and recovery from cytokine-mediated immunity by distinct immune cell types in a time-dependent manner. In lungs, the chemokine-driven response to viral invasion was highly elevated at 2 days post infection (dpi). In late infection, diseased lungs, post the innate immune process, showed recovery signs. The spleen established an even more immediate line of defense than the lungs, and the cytokine expression profile dropped at 7 dpi. At 5 dpi, spleen samples diverged into two distinct groups with different transcriptome profile and pathophysiology. Inhibition of consecutive host cell viral entry and massive immunoglobulin production and proteolysis inhibition seemed that one group endeavored to survive, while the other group struggled with developmental regeneration against consistent viral intrusion through the replication cycle. Our results may contribute to improved understanding of the longitudinal response to viral infection and development of potential therapeutics for hospitalized patients affected by SARS-CoV-2.

Severe Fever with Thrombocytopenia Syndrome Virus(SFTSV) 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.

The SARS-CoV-2 variant is rapidly spreading across the world and causes to resurge infections. We previously reported that CT-P59 presented its in vivo potency against Beta variants, despite its reduced activity in cell experiments. Yet, it remains uncertain to exert the antiviral effect of CT-P59 on Gamma, Delta and its associated variants (L452R). To tackle this question, we carried out cell tests and animal studies. CT-P59 showed neutralization against Gamma, Delta, Epsilon, and Kappa variants in cells, with reduced susceptibility. The mouse challenge experiments with Gamma and Delta variants substantiated in vivo potency of CT-P59 showing symptom remission and virus abrogation in the respiratory tract. Collectively, cell and animal studies showed that CT-P59 is effective against Gamma and Delta variants infection, hinting that CT-P59 has therapeutic potential for patients infected with Gamma, Delta and its associated variants.

A high-throughput, accurate screening is crucial for the prevention and control of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current methods, which involve sampling from the nasopharyngeal (NP) area by medical staffs, constitute a fundamental bottleneck in expanding the testing capacity. To meet the scales required for population-level surveillance, self-collectable specimens can be used; however, its low viral load has hindered its clinical adoption. Here, we describe a magnetic nanoparticle functionalized with synthetic apolipoprotein H (ApoH) peptides to capture, concentrate, and purify viruses. The ApoH assay demonstrates a viral enrichment efficiency of >90% for both SARS-CoV-2 and its variants, leading to an order of magnitude improvement in analytical sensitivity. For validation, we apply the assay to a total of 84 clinical specimens including nasal, oral, and mouth gargles obtained from COVID-19 patients. As a result, a 100% positivity rate is achieved from the patient-collected nasal and gargle samples, which exceeds that of the traditional NP swab method. The simple 12 min pre-enrichment assay enabling the use of self-collectable samples will be a practical solution to overcome the overwhelming diagnostic capacity. Furthermore, the methodology can easily be built on various clinical protocols, allowing its broad applicability to various disease diagnoses.

SARS-CoV-2, the coronavirus strain that initiated the COVID-19 pandemic, and its subsequent variants present challenges to vaccine development and treatment. As the coronavirus evades the host innate immune response at the initial stage of infection, the disease can have a long nonsymptomatic period. The uridylate-specific endoribonuclease Nsp15 processes the viral genome for replication and cleaves the polyU sequence in the viral RNA to interfere with the host immune system. This study screened natural compounds in vitro to identify inhibitors against Nsp15 from SARS-CoV-2. Three natural compounds, epigallocatechin gallate (EGCG), baicalin, and quercetin, were identified as potential inhibitors. Potent antiviral activity of EGCG was confirmed in plaque reduction neutralization tests with a SARS-CoV-2 strain (PRNT50 = 0.20 μM). Because the compound has been used as a functional food ingredient due to its beneficial health effects, we theorize that this natural compound may help inhibit viral replication while minimizing safety issues.

Introduction The world is currently fighting a COVID-19 pandemic, perhaps the most disruptive infectious disease outbreak since the 1918 Spanish influenza. Governments have taken drastic measures to curb the spread of SARS-CoV-2, and the development of safe and efficacious vaccine candidates is being accelerated. The possibility of vaccine-mediated disease enhancement with coronavirus vaccines has been flagged as a potential safety concern, and, despite the urgent need, should be thoroughly assessed as vaccines against SARS-CoV-2 are being tested. Area covered We review the in vivo evidence suggesting a theoretical risk of disease enhancement after vaccination with SARS-CoV and MERS-CoV vaccine candidates. We also identify knowledge gaps that need to be filled to maximize the chance of developing a safe vaccine and minimize the risk of encountering disease enhancement in vaccinated individuals after exposure to SARS-CoV-2. Expert opinion We compile and propose avenues to investigate the risk of vaccine-mediated disease enhancement both during pre-clinical and early clinical development. While the pressing need for a vaccine against COVID-19 (and future epidemic coronaviruses) cannot be ignored, we advocate to keep safety at the center of the debate. Protecting individuals with effective and safe vaccines should be a priority, even during extraordinary times like the COVID-19 pandemic.

The most widely used influenza vaccines are prepared by chemical inactivation. However, chemical, especially formalin, treatment-induced modifications of the antigenic structure of the virus are frequently associated with adverse effects including low efficacy of protection, unexpected immune responses, or exacerbation of disease. Gamma-irradiation was suggested as an alternative influenza virus inactivation method due to its great features of completely inactivating virus while not damaging the structures of protein antigens, and cross-protective ability against heterologous strains. However, immunological features of gamma radiation-inactivated influenza vaccine have not been fully understood. In this study, we aimed to investigate the humoral and cellular immune responses of gamma radiation-inactivated influenza vaccine. The gamma irradiation-inactivated influenza vaccine (RADVAX(FluA)) showed complete viral inactivation but retained normal viral structure with functional activities of viral protein antigens. Intranasal immunization of RADVAX(FluA) provided better protection against influenza virus infection than formalin-inactivated influenza virus (FIV) in mice. RADVAX(FluA) greatly enhanced the production of virus-specific serum IgG and alveolar mucosal IgA, which effectively neutralized HA (hemagglutinin) and NA (neuraminidase) activities, and blocked viral binding to the cells, respectively. Further analysis of IgG subclasses showed RADVAX(FluA)-immunized sera had higher levels of IgG1 and IgG2a than those of FIV-immunized sera. In addition, analysis of cellular immunity found RADVAX(FluA) induced strong dendritic cells (DC) activation resulting in higher DC-mediated activation of CD8(+) T cells than FIV. The results support improved immunogenicity by RADVAX(FluA).

P.1. or gamma variant also known as the Brazil variant, is one of the variants of concern (VOC) which appears to have high transmissibility and mortality. To explore the potency of the CT-P59 monoclonal antibody against P.1 variant, we tried to conduct binding affinity, in vitro neutralization, and in vivo animal tests. In in vitro assays revealed that CT-P59 is able to neutralize P.1 variant in spite of reduction in its binding affinity against a RBD (receptor binding domain) mutant protein including K417T/E484K/N501Y and neutralizing activity against P.1 pseudoviruses and live viruses. In contrast, in vivo hACE2 (human angiotensin-converting enzyme 2)-expressing TG (transgenic) mouse challenge experiment demonstrated that a clinically relevant or lower dosages of CT-P59 is capable of lowering viral loads in the respiratory tract and alleviates symptoms such as body weight losses and survival rates. Therefore, a clinical dosage of CT-P59 could compensate for reduced in vitro antiviral activity in P.1-infected mice, implying that CT-P59 has therapeutic potency for COVID-19 patients infected with P.1 variant.HighlightsCT-P59 could bind to and neutralize P.1 variant, but CT-P59 showed reduced susceptibility in in vitro tests.The clinical dosage of CT-P59 demonstrated in vivo therapeutic potency against P.1 variants in hACE2-expressing mice challenge study.CT-P59 ameliorates their body weight loss and prevents the lethality in P.1 variant-infected mice.

Highlights • RNA adjuvant was developed from the CrPV intergenic region IRES. • The RNA adjuvant functioned as an adjuvant with protein-based vaccines. • The RNA adjuvant increased vaccine efficacy and induced balanced Th1/Th2 response. • The RNA adjuvant enhanced APC chemotaxis., An ideal adjuvant should increase vaccine efficacy through balanced Th1/Th2 responses and be safe to use. Recombinant protein-based vaccines are usually formulated with aluminum (alum)-based adjuvants to ensure an adequate immune response. However, use of alum triggers a Th2-biased immune induction, and hence is not optimal. Although the adjuvanticity of RNA has been reported, a systematic and overall investigation on its efficacy is lacking. We found that single strand RNA (termed RNA adjuvant) derived from cricket paralysis virus intergenic region internal ribosome entry site induced the expression of various adjuvant-function-related genes, such as type 1 and 2 interferon (IFN) and toll-like receptor (TLR), T cell activation, and leukocyte chemotaxis in human peripheral blood mononuclear cells; furthermore, its innate and IFN transcriptome profile patterns were similar to those of a live-attenuated yellow fever vaccine. This suggests that protein-based vaccines formulated using RNA adjuvant function as live-attenuated vaccines. Application of the RNA adjuvant in mouse enhanced the efficacy of Middle East respiratory syndrome spike protein, a protein-subunit vaccine and human papillomavirus L1 protein, a virus-like particle vaccine, by activating innate immune response through TLR7 and enhancing pAPC chemotaxis, leading to a balanced Th1/Th2 responses. Moreover, the combination of alum and the RNA adjuvant synergistically induced humoral and cellular immune responses and endowed long-term immunity. Therefore, RNA adjuvants have broad applicability and can be used with all conventional vaccines to improve vaccine efficacy qualitatively and quantitively.

BackgroundWe investigated the safety and immunogenicity of two recombinant COVID-19 DNA vaccine candidates in first-in-human trials. GX-19 contains plasmid DNA encoding SARS-CoV-2 spike protein, and GX-19N contains plasmid DNA encoding SARS-CoV-2 receptor binding domain (RBD) foldon and nucleocapsid protein (NP) as well as plasmid DNA encoding SARS-CoV-2 spike protein.MethodsTwo open-label phase 1 trials of GX-19 and GX-19N safety and immunogenicity were performed in healthy adults aged 19–55 years. GX-19 trial participants received two vaccine injections (1·5 mg or 3·0 mg, 1:1 ratio) four weeks apart. GX-19N trial participants received two 3·0 mg vaccine injections four weeks apart.FindingsBetween June 17 and July 30 and December 28 and 31, 2020, 40 and 21 participants were enrolled in the GX-19 and GX-19N trials, respectively. Thirty-two participants (52·5%) reported 80 treatment-emergent adverse events (AE) after vaccination. All solicited AEs were mild except one case of moderate fatigue reported in the 1·5 mg GX-19 group. Binding antibody responses increased after vaccination in all groups. The geometric mean titers (GMTs) of spike-binding antibodies on day 57 were 85·74, 144·20, and 201·59 in the 1·5 mg, 3·0 mg GX-19 groups and the 3·0 mg GX-19N group, respectively. In GX-19N group, neutralizing antibody response (50% neutralizing titer using FRNT) significantly increased after vaccination, but GMT of neutralizing antibody on day 57 (37.26) was lower than those from human convalescent serum (288.78). GX-19N induced stronger T cell responses than GX-19. The magnitude of GX-19N-induced T cell responses was comparable to those observed in the convalescent PBMCs. GX-19N induced both SARS-CoV-2 spike- and NP-specific T cell responses, and the amino acid sequences of 15-mer peptides containing NP-specific T cell epitopes identified in GX-19N-vaccinated participants were identical with those of diverse SARS-CoV-2 variantsInterpretationGX-19N is safe, tolerated and induces humoral and broad SARS-CoV-2-specific T cell response which may enable cross-reactivity to emerging SARS-CoV-2 variants.FundingThis research was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (HQ20C0016, Republic of Korea).Research in contextEvidence before this studyTo overcome the COVID-19 outbreak, the development of safe and effective vaccines is crucial. Despite the successful clinical efficacy of the approved vaccines, concerns exist regarding emerging new SARS-CoV-2 variants that have mutated receptor binding domains in the spike protein. We searched PubMed for research articles published up to May 1, 2021, using various combinations of the terms “COVID-19” or “SARS-CoV-2”, “vaccine”, and “clinical trial”. No language or data restrictions were applied. We also searched theClinicalTrials.govregistry and World Health Organization (WHO) draft landscape of COVID-19 candidate vaccines for ongoing trials of COVID-19 vaccines up to May 1, 2021. Ten DNA-based vaccines, including the vaccine candidate reported here, are in ongoing clinical trials. Among these, safety and immunogenicity results were reported from only one phase 1 trial of a DNA vaccine against SARS-CoV-2 (INO-4800). INO-4800 demonstrated favorable safety and tolerability and was immunogenic, eliciting humoral and/or cellular immune responses in all vaccinated subjects. There is only one ongoing clinical trial of a vaccine against SARS-CoV-2 variants (mRNA-1273.351).Added value of this studyThis is the first-in-human phase 1 trial in healthy adults of a recombinant DNA vaccine for COVID-19 (GX-19N) containing the coding regions of both the spike and nucleocapsid proteins. This trial showed that GX-19N is safe, tolerated, and able to induce both humoral and cellular responses. A two-dose vaccination of 3·0 mg GX-19N (on days 1 and 29) induced significant humoral and cellular responses. The neutralizing geometric mean titers in individuals vaccinated with GX-19N were lower than those of human convalescent sera. However, the GX-19N group showed increased T cell responses, which was similar to those analyzed using convalescent PBMCs. Furthermore, GX-19N induced not only SARS-CoV-2 spike-specific T cell responses but also broad nucleocapsid-specific T cell responses, which were also specific to SARS-CoV-2 variants.Implications of all the available evidenceIt is important to note that GX-19N contains a plasmid encoding both the spike and nucleocapsid proteins, and that it showed broad SARS-CoV-2-specific T cell responses, which may allow cross-reactivity with emerging SARS-CoV-2 variants. Based on these safety and immunogenicity findings, GX-19N was selected for phase 2 immunogenicity trials.

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.

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., 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.

Highlights • Identification of neutralizing mAbs using MERS-CoV spike-pseudotyped virus. • Transmembrane domain-deleted spike subunit protein induced neutralizing antibodies. • Neutralizing antibodies could bind to RBD of MERS-CoV spike, but not vice versa. • Mutation in residue 506–509 or 529 of S elicits neutralization escape of MERS-CoV. • Our mAbs can be utilized for identification of specific mutation of MERS-CoV., 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.

Background: Cases of Middle East respiratory syndrome coronavirus (MERS-CoV) infection continue to rise in the Arabian Peninsula 7 years after it was first described in Saudi Arabia. MERS-CoV poses a significant risk to public health security because of an absence of currently available effective countermeasures. We aimed to assess the safety and immunogenicity of the candidate simian adenovirus-vectored vaccine expressing the full-length spike surface glycoprotein, ChAdOx1 MERS, in humans. Methods: This dose-escalation, open-label, non-randomised, uncontrolled, phase 1 trial was done at the Centre for Clinical Vaccinology and Tropical Medicine (Oxford, UK) and included healthy people aged 18–50 years with negative pre-vaccination tests for HIV antibodies, hepatitis B surface antigen, and hepatitis C antibodies (and a negative urinary pregnancy test for women). Participants received a single intramuscular injection of ChAdOx1 MERS at three different doses: the low-dose group received 5 × 109 viral particles, the intermediate-dose group received 2·5 × 1010 viral particles, and the high-dose group received 5 × 1010 viral particles. The primary objective was to assess safety and tolerability of ChAdOx1 MERS, measured by the occurrence of solicited, unsolicited, and serious adverse events after vaccination. The secondary objective was to assess the cellular and humoral immunogenicity of ChAdOx1 MERS, measured by interferon-γ-linked enzyme-linked immunospot, ELISA, and virus neutralising assays after vaccination. Participants were followed up for up to 12 months. This study is registered with ClinicalTrials.gov, NCT03399578. Findings: Between March 14 and Aug 15, 2018, 24 participants were enrolled: six were assigned to the low-dose group, nine to the intermediate-dose group, and nine to the high-dose group. All participants were available for follow-up at 6 months, but five (one in the low-dose group, one in the intermediate-dose group, and three in the high-dose group) were lost to follow-up at 12 months. A single dose of ChAdOx1 MERS was safe at doses up to 5 × 1010 viral particles with no vaccine-related serious adverse events reported by 12 months. One serious adverse event reported was deemed to be not related to ChAdOx1 MERS. 92 (74% [95% CI 66–81]) of 124 solicited adverse events were mild, 31 (25% [18–33]) were moderate, and all were self-limiting. Unsolicited adverse events in the 28 days following vaccination considered to be possibly, probably, or definitely related to ChAdOx1 MERS were predominantly mild in nature and resolved within the follow-up period of 12 months. The proportion of moderate and severe adverse events was significantly higher in the high-dose group than in the intermediate-dose group (relative risk 5·83 [95% CI 2·11–17·42], p Interpretation: ChAdOx1 MERS was safe and well tolerated at all tested doses. A single dose was able to elicit both humoral and cellular responses against MERS-CoV. The results of this first-in-human clinical trial support clinical development progression into field phase 1b and 2 trials.

SummaryThe unprecedented and rapid spread of SARS-CoV-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 primate seroconverted rapidly and exhibited 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 did not develop fever and reduced viral loads in contrast to non-vaccinated primates as a control. These findings indicate that GX-19 vaccination provides durable protective immune response and also support further development of GX-19 as a vaccine candidate for SARS-CoV-2 in human clinical trials.

There are several broadly neutralizing monoclonal antibodies that neutralize influenza viruses with different mechanisms from traditional polyclonal antibodies induced by vaccination. CT149, which is one of the broadly neutralizing antibodies, was also previously reported to neutralize group 2 and some of group 1 influenza viruses (13 out of 13 tested group 2 viruses and 5 out of 11 group 1 viruses). In this study, we developed another antibody with the aim of compensating partial coverage of CT149 against group 1 influenza viruses. CT120 was screened among different antibody candidates and mixed with CT149. Importantly, although the binding sites of CT120 and CT149 are close to each other, the two antibodies do not interfere. The mixture of CT120 and CT149, which we named as CT-P27, showed broad efficacy by neutralizing 37 viruses from 11 different subtypes, of both group 1 and 2 influenza A viruses. Moreover, CT-P27 showed in vivo therapeutic efficacy, long prophylactic potency, and synergistic effect with oseltamivir in influenza virus-challenged mouse models. Our findings provide a novel therapeutic opportunity for more efficient treatment of influenza.

Salmonella enterica serovar Typhi (S. Typhi) is a causative agent for typhoid fever and especially critical in developing countries. Although clinical studies for various typhoid conjugate vaccines (TCVs) have been performed, there are no comparative data on the immune responses of vaccines due to lack of harmonization of the serological assay. Recently, Typbar-TCV (Vi-TT) was prequalified by WHO and recommended for vaccination in endemic areas. Forty-eight serum samples were selected from a recent Vi-DT phase 1 study based on age cohort and anti-Vi IgG levels using an in-house ELISA. Anti-Vi IgG titers of 48 sera were also determined by Vacczyme ELISA, used in a Vi-TT phase 3 trial. A good correlation between the two assays was observed when the anti-Vi IgG titer was determined using Vacczyme ELISA based on the Vi-IgGR1,2011, U.S. reference reagent (Pearson correlation coefficient (r) = 0.991, P < 0.001) or Vacczyme ELISA calibrator (r = 0.991, P < 0.001). Based on the correlation, multiple linear regression model was developed to convert data of 281 sera (prior to vaccination and 28 days post first-dose) in the Vi-DT phase 1 study from in-house ELISA titers to Vacczyme ELISA values and then, compared with the Vi-TT results. Similar estimates of anti-Vi IgG GMT were observed after vaccination with the Vi-DT and Vi-TT vaccines [1626 EU/ml (95% CI: 1292–2047) vs 1293 EU/ml (95% CI: 1153–1449), respectively]. The method used here can be implemented to estimate and compare anti-Vi IgG levels between different clinical studies of TCVs. This approach enables comparison of the antibody responses among TCVs under development and may help facilitate licensing of new TCVs., Author summary Typhoid fever is an infectious and life-threatening disease in developing countries. Before 2017, Ty21a and Vi polysaccharide vaccines were licensed but these are not recommended in young children under 2-year-old. Vaccine manufacturers are developing typhoid Vi conjugate vaccines (TCVs) to improve immunogenicity. Typbar-TCV (Vi-TT) demonstrated its safety and immunogenicity in infants and recently, prequalified by WHO. Serum Vi-specific IgG antibody has been used to measure the immunogenicity of TCV in many clinical trials. However, due to lack of harmonized assay, comparison of the immunogenicity among various TCVs is not possible in the absence of head-to-head clinical trials. Recently, we evaluated immunogenicity of Vi-DT using an in-house ELISA in the clinical study. In this study, 48 sera were selected from Vi-DT phase 1 study and measured anti-Vi IgG using commercial Vacczyme ELISA kit, used in the phase 3 study of Vi-TT, to compare the immunogenicity between two vaccines. Based on the correlation between two assays, anti-Vi IgG of all participants in the Vi-DT study was converted to Vacczyme antibody value using statistical model and compared with results of Vi-TT phase 3 study. The antibody levels induced by two studies were similar in pre- and post-vaccinated sera. This approach enables to compare the antibody responses among TCVs under development and would facilitate licensing of new TCVs.

Developing effective mucosal subunit vaccine for the Streptococcus pneumoniae has been unsuccessful mainly because of their poor immunogenicity with insufficient memory T and B cell responses. We thus address whether such limitation can be overcome by introducing effective adjuvants that can enhance immunity and show here that polysorbitol transporter (PST) serves as a mucosal adjuvant for a subunit vaccine against the Streptococcus pneumoniae. Pneumococcal surface protein A (PspA) with PST adjuvant induced protective immunity against S. pneumoniae challenge, especially long-term T and B cell immune responses. Moreover, we found that the PST preferentially induced T helper (Th) responses toward Th2 or T follicular helper (Tfh) cells and, importantly, that the responses were mediated through antigen-presenting cells via activating a peroxisome proliferator-activated receptor gamma (PPAR-γ) pathway. Thus, these data indicate that PST can be used as an effective and safe mucosal vaccine adjuvant against S. pneumoniae infection. STATE OF SIGNIFICANCE: In this study, we suggested the nanoparticle forming adjuvant, PST works as an effective adjuvant for the pneumococcal vaccine, PspA. The PspA subunit vaccine together with PST adjuvant efficiently induced protective immunity, even in the long-term memory responses, against Streptococcus pneumoniae lethal challenge. We found that PspA with PST adjuvant induced dendritic cell activation followed by follicular helper T cell responses through PPAR-γ pathway resulting long-term memory antibody-producing cells. Consequently, in this paper, we suggest the mechanism for safe nanoparticle forming subunit vaccine adjuvant against pneumococcal infection.

Intramuscular (IM) vaccination with formalin-inactivated respiratory syncytial virus (FI-RSV) failed in clinical trials due to vaccine-enhanced respiratory disease. To test the efficacy of skin vaccination against respiratory syncytial virus (RSV), we investigated the immunogenicity, efficacy, and inflammatory disease after microneedle (MN) patch delivery of FI-RSV vaccine (FI-RSV MN) to the mouse skin with or without an adjuvant of monophosphoryl lipid A (MPL). Compared to IM vaccination, MN patch delivery of FI-RSV was more effective in clearing lung viral loads and preventing weight loss, and in diminishing inflammation, infiltrating immune cells, and T helper type 2 (Th2) CD4 T cell responses after RSV challenge. With MPL adjuvant, MN patch delivery of FI-RSV significantly increased the immunogenicity and efficacy as well as preventing RSV disease as evidenced by lung viral clearance and avoiding pulmonary histopathology. Improved efficacy and prevention of disease by FI-RSV MN with MPL were correlated with no sign of airway resistance, lower levels of Th2 cytokines and infiltrating innate inflammatory cells, and higher levels of Th1 T cell responses into the lung. This study suggests that MN patch delivery of RSV vaccines to the skin with MPL adjuvant would be a promising vaccination method.

Shigella is a highly prevalent bacterium causing acute diarrhea and dysentery in developing countries. Shigella infections are treated with antibiotics but Shigellae are increasingly resistant to these drugs. Vaccination can be a countermeasure against emerging antibiotic-resistant shigellosis. Because of the structural variability in Shigellae O-antigen polysaccharides (Oag), cross-protective Shigella vaccines cannot be derived from single serotype-specific Oag. We created an attenuated Shigella flexneri 2a strain with one rather than multiple Oag units by disrupting the Oag polymerase gene (Δwzy), which broadened protective immunogenicity by exposing conserved surface proteins. Inactivated Δwzy mutant cells combined with Escherichia coli double mutant LT(R192G/L211A) as adjuvant, induced potent antibody responses to outer membrane protein PSSP-1, and type III secretion system proteins IpaB and IpaC. Intranasal immunization with the vaccine preparation elicited cross-protective immunity against S. flexneri 2a, S. flexneri 3a, S. flexneri 6, and Shigella sonnei in a mouse pneumonia model. Thus, S. flexneri 2a Δwzy represents a promising candidate strain for a universal Shigella vaccine.

Background: Assessing immune response after rotavirus vaccination consists in measuring serum or plasma IgA and IgG antibodies, but these assays provide very little information about the mucosal immune response. Thus the development of assays for detection of mucosal immune response following rotavirus vaccination is essential. We evaluate to assess circulating antibody-secreting cells (ASCs) as a potential means to evaluate mucosal immune responses to rotavirus vaccine. Methods: 372 subjects, aged 6 weeks, were enrolled in the study. All the subjects were assigned to receive two doses of Rotarix® vaccine. Using a micro-modified whole blood-based ELISPOT assay, circulating rotavirus type-specific IgA- and IgG-ASCs, including gut homing β7+ ASCs, were enumerated on week 6 before the first dose of Rotarix vaccination at 7 weeks of age and week 18 after the second vaccination at 17 weeks of age. Plasma samples collected before vaccination, and after two doses of Rotarix® vaccination were tested for plasma rotavirus IgA titers. Results: Two doses of Rotarix® provided to induce sero-protective titer of ≥ 20 Units in 35% of subjects. Total blood IgA- ASC responses were detected in 26.4% of subjects who were non-responder before vaccination. Among responders, 47% of the subjects also have sero-protective plasma IgA titers. Discussion: Our results suggest that virus-specific blood gut homing ASCs were detected and provide insight into mucosal immune response after rotavirus vaccination. Further studies are needed to evaluate the duration of such immune responses and to assess the programmatic utility of this whole blood-based mucosal ASC testing for the rotavirus immunization program.

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 vivocytotoxic 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.

During influenza pandemics, secondary pneumococcal infections cause excessive mortality. However, the current 13-valent pneumococcal conjugate vaccine, PCV13, provides only limited protection against secondary infection. Therefore, a more effective pneumococcal vaccine is required to protect against secondary pneumococcal infections. Here, intranasal immunization with an attenuated pneumococcal pep27 mutant provides protection from influenza virus infection, and also from secondary pneumococcal challenge. These results indicate that mucosal immunity might be an effective way to reduce the morbidity and mortality due to secondary pneumococcal infections during influenza pandemics.

Respiratory syncytial virus (RSV) is a major viral pathogen that causes severe lower respiratory tract infections in infants and the elderly worldwide. Infants with severe RSV bronchiolitis tend to experience more wheezing and asthma in later childhood. Because invariant natural killer T (iNKT) cells are associated with the asthma pathology, we investigated whether neonatal iNKT cells are involved in the aggravation of pulmonary diseases following RSV infection in mice. Intranasal exposure to the iNKT cell ligand alpha-galactosylceramide (alpha-GC) with RSV primary infection in neonatal mice elicited neither cytokine production (except for a slight increase of IL-5) nor pulmonary eosinophilia, despite the presence of both CD1d(+) cells and NKT cells. Interestingly, in adult mice re-infected with RSV, neonatal iNKT cell sensitization by alpha-GC during RSV primary infection resulted in much higher levels of pulmonary Th2 cytokines and elevated eosinophilia with airway hyperresponsiveness, whereas this was not observed in cd1d knockout mice. In contrast, alpha-GC priming of adults during RSV re-infection did not induce more severe airway symptoms than RSV re-infection in the absence of alpha-GC. alpha-GC co-administration during RSV primary infection facilitated RSV clearance regardless of age, but viral clearance following re-infection was not iNKT cell-dependent. This study clearly demonstrates that RSV-induced immune responses can be altered by iNKT cells, suggesting that neonatal iNKT cell sensitization during RSV primary infection is associated with exacerbation of pulmonary diseases following RSV re-infection in adulthood.

Serum vibriocidal antibody assays have long been used to evaluate the immunogenicity of cholera vaccines formulated with killed whole-cell Vibrio cholerae. However, the antibody isotypes responsible for the serum vibriocidal activity are not fully characterized. In this study, we examined 20 clinical serum samples obtained from human subjects who had been vaccinated with a killed, whole-cell cholera vaccine and a positive control, human convalescent sera with high vibriocidal activity, to determine which isotype antibody is associated with the vibriocidal activity. Antibody isotypes from pooled convalescent sera were fractionated by size-exclusion column chromatography, and the major vibriocidal activity was detected in the IgM fraction. Depletion of IgM antibodies in the convalescent sera produced a significant (P