Human parainfluenza virus 3 vaccine candidates attenuated by codon-pair deoptimization are immunogenic and protective in hamsters.

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

African swine fever virus I73R is a critical virulence-related gene: A potential target for attenuation.

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal disease in pigs, posing a threat to the global pig industry. Whereas some ASFV proteins have been found to play important roles in ASFV-host interaction, the functional roles of many proteins are still largely unknown. In this study, we identified I73R, an early viral gene in the replication cycle of ASFV, as a key virulence factor. Our findings demonstrate that pI73R suppresses the host innate immune response by broadly inhibiting the synthesis of host proteins, including antiviral proteins. Crystallization and structural characterization results suggest that pI73R is a nucleic-acid-binding protein containing a Zα domain. It localizes in the nucleus and inhibits host protein synthesis by suppressing the nuclear export of cellular messenger RNA (mRNAs). While pI73R promotes viral replication, the deletion of the gene showed that it is a nonessential gene for virus replication. In vivo safety and immunogenicity evaluation results demonstrate that the deletion mutant ASFV-GZΔI73R is completely nonpathogenic and provides effective protection to pigs against wild-type ASFV. These results reveal I73R as a virulence-related gene critical for ASFV pathogenesis and suggest that it is a potential target for virus attenuation. Accordingly, the deletion mutant ASFV-GZΔI73R can be a potent live-attenuated vaccine candidate.

Rational design of self-amplifying virus-like vesicles with Ebola virus glycoprotein as vaccines.

As emerging and re-emerging pathogens, filoviruses, especially Ebola virus (EBOV), pose a great threat to public health and require sustained attention and ongoing surveillance. More vaccines and antiviral drugs are imperative to be developed and stockpiled to respond to unpredictable outbreaks. Virus-like vesicles, generated by alphavirus replicons expressing homogeneous or heterogeneous glycoproteins (GPs), have demonstrated the capacity of self-propagation and shown great potential in vaccine development. Here, we describe a novel class of EBOV-like vesicles (eVLVs) incorporating both EBOV GP and VP40. The eVLVs exhibited similar antigenicity as EBOV. In murine models, eVLVs were highly attenuated and elicited robust GP-specific antibodies with neutralizing activities. Importantly, a single dose of eVLVs conferred complete protection in a surrogate EBOV lethal mouse model. Furthermore, our VLVs strategy was also successfully applied to Marburg virus (MARV), the representative member of the genus Marburgvirus. Taken together, our findings indicate the feasibility of an alphavirus-derived VLVs strategy in combating infection of filoviruses represented by EBOV and MARV, which provides further evidence of the potential of this platform for universal live-attenuated vaccine development.

Assessing clinical benefits of live-attenuated vaccination in post-liver transplant patients: Analysis of breakthrough infections and natural boosters.

Recently, live-attenuated measles, rubella, varicella, and mumps vaccines have been administered to carefully selected post-liver transplant patients. Although attention has been focused on post-vaccination antibody titers and adverse events, the real-life clinical benefits remain unclear. A comprehensive analysis of breakthrough infections and natural boosters (asymptomatic cases with significant elevation in virus antibody titers) following immunization post-liver transplantation was conducted from 2002-2023, exploring the timing, frequency, correlation with domestic outbreaks, and degree of antibody elevation. During the median 10-year observation period among 68 post-liver transplant patients, breakthrough infections occurred only in chickenpox, with 7 mild cases (1 episode/64 person-years). A total of 59 natural booster episodes (1, 5, 20, and 33 for measles, rubella, chickenpox, and mumps, respectively) were observed, with incidence rates of 1 per 569, 110, 22, and 17 person-years, respectively. The timing of natural boosters closely correlated with domestic outbreaks (P < .05 in chickenpox and mumps), influenced by local vaccine coverage. The degree of antibody elevation was significantly higher in individuals with breakthrough infections than in those with natural boosters (P < .05). These findings suggest that immunization with live-attenuated vaccines for post-liver transplant patients has demonstrated clinical benefits. Furthermore, mass vaccination has a positive impact on post-transplant patient outcomes.

Deletion of DP148R, DP71L, and DP96R Attenuates African Swine Fever Virus, and the Mutant Strain Confers Complete Protection against Homologous Challenges in Pigs.

The African swine fever virus (ASFV) has caused a devastating pandemic in domestic and wild swine, causing economic losses to the global swine industry. Recombinant live attenuated vaccines are an attractive option for ASFV treatment. However, safe and effective vaccines against ASFV are still scarce, and more high-quality experimental vaccine strains need to be developed. In this study, we revealed that deletion of the ASFV genes DP148R, DP71L, and DP96R from the highly virulent isolate ASFV CN/GS/2018 (ASFV-GS) substantially attenuated virulence in swine. Pigs infected with 104 50% hemadsorbing doses of the virus with these gene deletions remained healthy during the 19-day observation period. No ASFV infection was detected in contact pigs under the experimental conditions. Importantly, the inoculated pigs were protected against homologous challenges. Additionally, RNA sequence analysis showed that deletion of these viral genes induced significant upregulation of the host histone H3.1 gene (H3.1) and downregulation of the ASFV MGF110-7L gene. Knocking down the expression of H3.1 resulted in high levels of ASFV replication in primary porcine macrophages in vitro. These findings indicate that the deletion mutant virus ASFV-GS-Δ18R/NL/UK is a novel potential live attenuated vaccine candidate and one of the few experimental vaccine strains reported to induce full protection against the highly virulent ASFV-GS virus strain. IMPORTANCE Ongoing outbreaks of African swine fever (ASF) have considerably damaged the pig industry in affected countries. Thus, a safe and effective vaccine is important to control African swine fever spread. Here, an ASFV strain with three gene deletions was developed by knocking out the viral genes DP148R (MGF360-18R), NL (DP71L), and UK (DP96R). The results showed that the recombinant virus was completely attenuated in pigs and provided strong protection against parental virus challenge. Additionally, no viral genomes were detected in the sera of pigs housed with animals infected with the deletion mutant. Furthermore, transcriptome sequencing (RNA-seq) analysis revealed significant upregulation of histone H3.1 in virus-infected macrophage cultures and downregulation of the ASFV MGF110-7L gene after viral DP148R, UK, and NL deletion. Our study provides a valuable live attenuated vaccine candidate and potential gene targets for developing strategies for anti-ASFV treatment.

Molecular Engineering of a Mammarenavirus with Unbreachable Attenuation.

Several mammarenaviruses cause severe hemorrhagic fever (HF) disease in humans and pose important public health problems in their regions of endemicity. There are no United States (US) Food and Drug Administration (FDA)-approved mammarenavirus vaccines, and current anti-mammarenavirus therapy is limited to an off-label use of ribavirin that has limited efficacy. Mammarenaviruses are enveloped viruses with a bi-segmented negative-strand RNA genome. Each genome segment contains two open reading frames (ORF) separated by a noncoding intergenic region (IGR). The large (L) segment encodes the RNA dependent RNA polymerase, L protein, and the Z matrix protein, whereas the small (S) segment encodes the surface glycoprotein precursor (GPC) and nucleoprotein (NP). In the present study, we document the generation of a recombinant form of the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) expressing a codon deoptimized (CD) GPC and containing the IGR of the S segment in both the S and L segments (rLCMV/IGR-CD). We show that rLCMV/IGR-CD is fully attenuated in C57BL/6 (B6) mice but able to provide complete protection upon a single administration against a lethal challenge with LCMV. Importantly, rLCMV/IGR-CD exhibited an unbreachable attenuation for its safe implementation as a live-attenuated vaccine (LAV). IMPORTANCE Several mammarenaviruses cause severe disease in humans and pose important public health problems in their regions of endemicity. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenaviral therapy is limited to an off-label use of ribavirin whose efficacy is controversial. Here, we describe the generation of recombinant version of the prototypic mammarenavirus lymphocytic choriomeningitis virus (rLCMV) combining the features of a codon deoptimized (CD) GPC and the noncoding intergenic region (IGR) of the S segment in both S and L genome segments, called rLCMV/IGR-CD. We present evidence that rLCMV/IGR-CD has excellent safety and protective efficacy features as live-attenuated vaccine (LAV). Importantly, rLCMV/IGR-CD prevents, in coinfected mice, the generation of LCMV reassortants with increased virulence. Our findings document a well-defined molecular strategy for the generation of mammarenavirus LAV candidates able to trigger long-term protective immunity, upon a single immunization, while exhibiting unique enhanced safety features, including unbreachable attenuation.

Repurposing of Zika virus live-attenuated vaccine (ZIKV-LAV) strains as oncolytic viruses targeting human glioblastoma multiforme cells.

Glioblastoma multiforme (GBM) is the most common malignant primary brain cancer affecting the adult population. Median overall survival for GBM patients is poor (15 months), primarily due to high rates of tumour recurrence and the paucity of treatment options. Oncolytic virotherapy is a promising treatment alternative for GBM patients, where engineered viruses selectively infect and eradicate cancer cells by inducing cell lysis and eliciting robust anti-tumour immune response. In this study, we evaluated the oncolytic potency of live-attenuated vaccine strains of Zika virus (ZIKV-LAV) against human GBM cells in vitro. Our findings revealed that Axl and integrin αvß5 function as cellular receptors mediating ZIKV-LAV infection in GBM cells. ZIKV-LAV strains productively infected and lysed human GBM cells but not primary endothelia and terminally differentiated neurons. Upon infection, ZIKV-LAV mediated GBM cell death via apoptosis and pyroptosis. This is the first in-depth molecular dissection of how oncolytic ZIKV infects and induces death in tumour cells.

Δfur mutant as a potential live attenuated vaccine (LAV) candidate protects American eels (Anguilla rostrata) from Vibrio harveyi infection.

The eel farming industry is highly susceptible to Vibriosis. Although various types of vaccines against Vibriosis have been investigated, there is limited research on decreasing the virulence of Vibrions through gene knockout and utilizing it as live attenuated vaccines (LAV). In this study, we aim to develop a LAV candidate against Vibrio harveyi infection in American eels (Anguilla rostrata) using a ferric uptake regulator (fur) gene mutant strain of V. harveyi (Δfur mutant). After the eels were administrated with the Δfur mutant at the dose of 4 × 102 cfu/g body weight, the phagocytic activity of the leucocytes, plasma IgM antibody titers, activity of lysozyme and Superoxide Dismutase (SOD) enzyme, and gene expression levels of 18 immune related proteins were detected to evaluate the protection effect of the LAV. Preliminary findings suggest that the LAV achieved over 60% relative percent survival (RPS) after the American eels were challenged by a wild-type strain of V. harveyi infection on 28 and 42 days post the immunization (dpi). The protection was mainly attributed to increased plasma IgM antibody titers, higher levels of lysozyme, enhanced activity of SOD and some regulated genes encoded immune related proteins. Together, the Δfur mutant strain of V. harveyi, as a novel LAV vaccine, demonstrates promising protective effects against V. harveyi infection in American eels, thus presenting a potential candidate vaccine for fish farming.

Early divergent responses to virulent and attenuated vaccine isolates of Flavobacterium covae sp. nov. In channel catfish, Ictalurus punctatus.

Columnaris disease continues to inflict substantial losses among freshwater cultured species since its first description one hundred years ago. The experimental and anecdotal evidence suggests an expanded range and rising virulence of columnaris worldwide due to the warming global climate. The channel catfish (Ictalurus punctatus) are particularly vulnerable to columnaris. A recently developed live attenuated vaccine (17-23) for Flavobacterium columnare (now Flavobacterium covae sp. nov.) demonstrated superior protection for vaccinated catfish against genetically diverse columnaris isolates. In this study, we aimed to elucidate the molecular mechanisms and patterns of immune evasion and host manipulation linked to virulence by comparing gene expression changes in the host after the challenge with a virulent (BGSF-27) or live attenuated F. covae sp. nov. vaccine (17-23). Thirty-day-old fry were accordingly challenged with either virulent or vaccine isolates. Gill tissues were collected at 0 h (control), 1 h, and 2 h post-infection, which are two critical time points in early host-pathogen interactions. Transcriptome profiling of the gill tissues revealed a larger number (518) of differentially expressed genes (DEGs) in vaccine-exposed fish than those exposed to the virulent pathogen (321). Pathway analyses suggested potent suppression of early host immune responses by the virulent isolate through a higher expression of nuclear receptor corepressors (NCoR) responsible for antagonizing macrophage and T-cell signaling. Conversely, in vaccinated fry, we observed induction of Ca2+/calmodulin-dependent protein kinase II (CAMKII), responsible for clearing NCoR, and commensurate up-regulation of transcription factor AP-1 subunits, c-Fos, and c-Jun. As in mammalian systems, AP-1 expression was connected with a broad immune activation in vaccinated fry, including induction of CC chemokines, proteinases, iNOS, and IL-12b. Relatedly, divergent expression patterns of Src tyrosine kinase Lck, CD44, and CD28 indicated a delay or suppression of T-cell adhesion and activation in fry exposed to the virulent isolate. Broader implications of these findings will be discussed. The transcriptomic differences between virulent and attenuated bacteria may offer insights into how the host responds to the vaccination or infection and provide valuable knowledge to understand the early immune mechanisms of columnaris disease in aquaculture.

Construction and characterization of Aeromonas hydrophila crp and fur deletion mutants and evaluation of its potential as live-attenuated vaccines in crucian carp.

Aeromonas hydrophila (A. hydrophila) is a typical zoonotic pathogenic bacterium that infects humans, animals, and fish. It has been reported that the Fur, a Fe2+ regulatory protein, and the Crp, a cAMP receptor protein, play important roles in bacterial virulence in many bacteria, but no research has been investigated on A. hydrophila. In this study, the Δfur and Δcrp mutant strains were constructed by the suicide plasmid method. These two mutant strains exhibited a slightly diminished bacterial growth and also were observed some alterations in the number of outer membrane proteins, and the disappearance of hemolysis in the Δcrp strain. Animal experiments of crucian carp showed that the Δfur and Δcrp mutant strains significantly decreased virulence compared to the wild-type strain, and both mutant strains were able to induce good immune responses by two kinds of administration routes of intraperitoneal immunization (i.p) and immersion immunization, and the protection rates through intraperitoneal injection of Δfur and Δcrp to crucian carp were as high as 83.3 % and 73.3 %, respectively, and immersion immunization route of Δfur and Δcrp to crucian carp provided protection as high as 40 % and 20 %, respectively. These two mutant strains showed abilities to induce changes in enzymatic activities of the non-specific enzymes SOD, LZM, AKP, and ACP in crucian carp. Together, these results indicated the Δfur and Δcrp mutants were safe and effective candidate vaccine strains, showing good protection against the wild-type A. hydrophila challenge.

The role of aroA and ppk1 in Aeromonas veronii pathogenicity and the efficacy evaluation of mutant strain AV-ΔaroA/ppk1 as a live attenuated vaccine.

Aeromonas veronii is an opportunistic pathogen that poses great threat to aquaculture and human health, so there is an urgent need for green and efficient methods to deal with its infection. In this study, single and double gene deletion strains (AV-ΔaroA, AV-Δppk1 and AV-ΔaroA/ppk1) that can be stably inherited were constructed. Pathogenicity test showed that the toxicity of AV-ΔaroA and AV-ΔaroA/ppk1 was significantly lower compared to wild-type A. veronii. Biological characterization analysis revealed that the decrease in pathogenicity might be due to the declined growth, motility, biofilm formation abilities and the expression of virulence-related genes in mutants. Subsequently, we evaluated the efficacy of AV-ΔaroA/ppk1 as a live attenuated vaccine (LAV). Safety assessment experiments showed that AV-ΔaroA/ppk1 injected at a concentration of 3 × 107 CFU/mL was safe for C. carassius. The relative percentage survival of AV-ΔaroA/ppk1 was 67.85 %, significantly higher than that of the inactivated A. veronii, which had an RPS of 54.84 %. This improved protective effect was mainly attributed to the increased levels of A. veronii specific IgM antibody, enhanced alkaline phosphatase, lysozyme and superoxide dismutase activities, as well as higher expression levels of several immune related genes. Together, these findings deepen our understanding of the functional roles of aroA and ppk1 in A. veronii pathogenicity, provide a good candidate of LAV for A. veronii.

Cell-mediated and humoral immune responses of cyprinids induced by a live attenuated vaccine against cyprinid herpesvirus 2 infection in comparison to the virus non-permissive high temperature water treatment.

The live attenuated vaccine (P7-P8 strain) against cyprinid herpesvirus 2 (CyHV-2) infection of goldfish shows high protective efficacy. However, the underlying immune mechanism induced by P7-P8 vaccination remains unknown. It is known that the fish survived in the primary infection with CyHV-2 by the virus non-permissive high temperature (HT) water treatment elicit immunity against secondary virus challenge. In this study, the immunity induced by the P7-P8 vaccine was compared with that by HT treatment. To further explore the immunological responses of cyprinids, in addition to goldfish Carassius auratus, susceptible isogenic ginbuna C. auratus langsdorfii was included in this study. In the primary immune response, cyprinids were vaccinated with P7-P8, or treated with HT. In the secondary immune response, cyprinids were challenged with the virulent CyHV-2. The percentage dynamics of CD4-1 and CD8α positive lymphocytes were determined during the primary and secondary immune responses of the two cyprinids. Blood plasma was sampled to assess the anti-CyHV-2 IgM antibody titers. The vaccination with P7-P8 and HT-treatment induced high protection immunity in the cyprinids with relative percentage survival of over 88 % against virulent virus challenge. Our finding shows that the CD8α positive lymphocytes rather than CD4-1 positive lymphocytes play an important role in the secondary immune responses of cyprinids vaccinated with P7-P8. The CD4-1 positive lymphocytes rather than CD8α positive lymphocytes play an important role in the secondary immune responses of cyprinids treated with HT. The antibody titer of vaccinated cyprinids did not increase greatly even after virulent virus challenge. The results suggest the vaccine activates the CD8α cells and the secondary cell-mediated immunity. The differences in the induced immunity mechanisms in fish by the two measures might be based on the virus either being an avirulent virus form that cannot evade host responses or a virulent virus form that cannot propagate at non-permissive temperature.

Effect of temperature on the protective efficacy of a live attenuated vaccine against herpesviral haematopoietic necrosis in goldfish.

The live attenuated vaccine P7-P8 strain against herpesviral haematopoietic necrosis, which is caused by cyprinid herpesvirus 2 (CyHV-2), exhibits high protective efficacy in goldfish at 25°C, the predominant temperature for this disease; however, the effect of water temperature during the vaccination period on efficacy has not been determined. In this study, an in vitro experiment revealed that the vaccine strain grew between 15 and 30°C in the goldfish cell line RyuF-2. Subsequent in vivo efficacy tests were conducted with vaccination temperatures ranging from 15 to 30°C. During the vaccination period, organs were sampled to determine the vaccine growth dynamics. Blood plasma was collected to assess anti-CyHV-2 antibody titres. The protective efficacy of the vaccine at 15, 20, 25, and 30°C after subsequent virulent CyHV-2 challenge resulted in a relative percentage survival of 73.3%, 77.8%, 100%, and 77.8%, respectively, which indicated that the vaccine is effective over this temperature range. The vaccine virus load in the spleen was lowest at 15°C (103.7 DNA copies/mg) and highest at 25°C (106.5 DNA copies/mg). This indicates that the vaccine virus load over 104 DNA copies/mg may elicit sufficient acquired immunity. No significant differences in antibody titre were observed between groups, which suggests that cell-mediated immunity can be fundamentally involved in protection.

Internalization of rabies virus glycoprotein differs between pathogenic and attenuated virus strains.

The zoonotic rabies virus (RABV) is a non-segmented negative-sense RNA virus classified within the family Rhabdoviridae, and is the most common aetiological agent responsible for fatal rabies disease. The RABV glycoprotein (G) forms trimeric spikes that protrude from RABV virions and mediate virus attachment, entry and spread, and is a major determinant of RABV pathogenesis. A range of RABV strains exist that are highly pathogenic in part due to their ability to evade host immune detection. However, some strains are disease-attenuated and can be cleared by host defences. A detailed molecular understanding of how strain variation relates to pathogenesis is currently lacking. Here, we reveal key differences in the trafficking profiles of RABV-G proteins from the challenge virus standard strain (CVS-11) and a highly attenuated vaccine strain SAD-B19 (SAD). We show that CVS-G traffics to the cell surface and undergoes rapid internalization through both clathrin- and cholesterol-dependent endocytic pathways. In contrast, SAD-G remains resident at the plasma membrane and internalizes at a significantly slower rate. Through engineering hybrids of CVS-G and SAD-G, we show that the cytoplasmic tail of CVS-G is the key determinant of these different internalization profiles. Alanine scanning further revealed that mutation of Y497 in CVS-G (H497 in SAD-G) could reduce the rate of internalization to SAD-G levels. Together, these data reveal new phenotypic differences between CVS-G and SAD-G proteins that may contribute to altered in vivo pathogenicity.

Multiple Immune Defects in Two Patients with Novel DOCK2 Mutations Result in Recurrent Multiple Infection Including Live Attenuated Virus Vaccine.

The dedicator of cytokinesis 2(DOCK2) protein, an atypical guanine nucleotide exchange factor (GEFs), is a member of the DOCKA protein subfamily. DOCK2 protein deficiency is characterized by early-onset lymphopenia, recurrent infections, and lymphocyte dysfunction, which was classified as combined immune deficiency with neutrophil abnormalities as well. The only cure is hematopoietic stem cell transplantation. Here, we report two patients harboring four novel DOCK2 mutations associated with recurrent infections including live attenuated vaccine-related infections. The patient's condition was partially alleviated by symptomatic treatment or intravenous immunoglobulin. We also confirmed defects in thymic T cell output and T cell proliferation, as well as aberrant skewing of T/B cell subset TCR-Vß repertoires. In addition, we noted neutrophil defects, the weakening of actin polymerization, and BCR internalization under TCR/BCR activation. Finally, we found that the DOCK2 protein affected antibody affinity although with normal total serum immunoglobulin. The results reported herein expand the clinical phenotype, the pathogenic DOCK2 mutation database, and the immune characteristics of DOCK2-deficient patients.

Optimizing the Live Attenuated Influenza A Vaccine Backbone for High-Risk Patient Groups.

Together with inactivated influenza vaccines (IIV), live attenuated influenza vaccines (LAIV) are an important tool to prevent influenza A virus (IAV) illnesses in patients. LAIVs present the advantages to have a needle-free administration and to trigger a mucosal immune response. LAIV is approved for healthy 2- to 49-year old individuals. However, due to its replicative nature and higher rate of adverse events at-risk populations are excluded from the benefits of this vaccine. Using targeted mutagenesis, we modified the nonstructural protein 1 of the currently licensed LAIV in order to impair its ability to bind the host cellular protein CPSF30 and thus its ability to inhibit host mRNA poly-adenylation. We characterized our optimized LAIV (optiLAIV) in three different mouse models mimicking healthy and high-risk patients. Using a neonatal mouse model, we show faster clearance of our optimized vaccine compared to the licensed LAIV. Despite lower replication, optiLAIV equally protected mice against homosubtypic and hetesubtypic influenza strain challenges. We confirmed the safer profile of optiLAIV in Stat1-/- mice (highly susceptible to viral infections) by showing no signs of morbidity compared to a 50% mortality rate observed following LAIV inoculation. Using a human nasal 3D tissue model, we showed an increased induction of ER stress-related genes following immunization with optiLAIV. Induction of ER stress was previously shown to improve antigen-specific immune responses and is proposed as the mechanism of action of the licensed adjuvant AS03. This study characterizes a safer LAIV candidate in two mouse models mimicking infants and severely immunocompromised patients and proposes a simple attenuation strategy that could broaden LAIV application and reduce influenza burden in high-risk populations. IMPORTANCE Live attenuated influenza vaccine (LAIV) is a needle-free, mucosal vaccine approved for healthy 2- to 49-year old individuals. Its replicative nature and higher rate of adverse events excludes at-risk populations. We propose a strategy to improve LAIV safety and explore the possibility to expand its applications in children under 2-year old and immunocompromised patients. Using a neonatal mouse model, we show faster clearance of our optimized vaccine (optiLAIV) compared to the licensed LAIV. Despite lower replication, optiLAIV equally protected mice against influenza virus challenges. We confirmed the safer profile of optiLAIV in Stat1-/- mice (highly susceptible to viral infections) by showing no signs of morbidity compared to a 50% mortality rate from LAIV. OptiLAIV could expand the applications of the current LAIV and help mitigate the burden of IAV in susceptible populations.

A booster administration of the OKA/SK strain causes fatal disseminated varicella in an immunocompetent child.

Live varicella vaccines are known to provide robust immunity against varicella zoster virus (VZV) infections. However, problems with viral attenuation have led to pathogenic VZV vaccine strains causing varicella-like rash and herpes zoster in immunocompetent children after immunization. We report the first fatal case of VZV infection caused by OKA/SK strain contained in the vaccine administrated as a booster shot in an immunocompetent child, which has been independently developed from any currently available varicella vaccines that are OKA strain or MAV/06 strain based. The patient died due to sudden pulmonary alveolar hemorrhage as a secondary complication of VZV pneumonitis. Sequencing of the four SNPs unique to the OKA/SK strain (SNP loci 14 035T; 32 626C; 58 777G; 70 319G) enabled discrimination of the strain responsible for the disseminated infection. OKA/SK strain does not have any SNPs in ORF62 postulated to be responsible for the attenuation of varicella vaccines which have been safely and effectively used world-wide or locally, and exclusively enriches a virulent factor in ORF31 identified in parental OKA strain, thus possibly resulting in disseminated VZV infection leading to mortality. Therefore, actions need to be taken to prevent vaccine related morbidity and mortality in children.

A live attenuated DIVA vaccine affords protection against Listeria monocytogenes challenge in sheep.

Listeria monocytogenes (Lm) is a deadly foodborne pathogen that comprises 14 serotypes, among which, serotype 4b Lm is the primary cause of listeriosis outbreaks in humans and animals. Here, we evaluated the safety, immunogenicity, and protective efficacy of a serotype 4b vaccine candidate Lm NTSNΔactA/plcB/orfX in sheep. The infection dynamics, clinical features, and pathological observation verified that the triple genes deletion strain has adequate safety for sheep. Moreover, NTSNΔactA/plcB/orfX significantly stimulated humoral immune response and provided 78% immune protection to sheep against lethal wild-type strain challenge. Notably, the attenuated vaccine candidate could differentiate infected and vaccinated animals (DIVA) via serology determination of the antibody against listeriolysin O (LLO, encoded by hly) and phosphatidylinositol-specific phospholipase C (PI-PLC, encoded by plcB). These data suggest that the serotype 4b vaccine candidate has high efficacy, safety, and DIVA characteristics, and may be used to prevent Lm infection in sheep. Our study provides a theoretical basis for its future application in livestock and poultry breeding.

A single-dose strategy for immunization with live attenuated vaccines is an effective option before treatment initiation in multiple sclerosis patients.

BACKGROUND: Mumps-Measles-Rubella (MMR) and Varicella zoster vaccines (VAR) are live attenuated vaccines, usually administered in a two-dose scheme at least 4 weeks apart. However, single-dose immunization schemes may also be effective and can reduce delays in immunosuppressive treatment initiation in patients with multiple sclerosis (pwMS) who need to be immunized. OBJECTIVES: To evaluate the immunogenicity of a single-dose attempt (SDA) versus the standard immunization scheme (SIS) with VAR and/or MMR in pwMS. METHODS: Retrospective observational study in pwMS vaccinated against VAR and/or MMR. We compared seroprotection rates and antibody geometric mean titers (GMTs) between the two strategies. RESULTS: Ninety-six patients were included. Thirty-one patients received VAR and 67 MMR. In the SDA group, the seroprotection rate was 66.7% (95% confidence interval (CI): 53.3-78.3) versus 97.2% (95% CI: 85.5-99.9) in the SIS (p < 0.001). For the seroprotected patients, GMTs were similar for both schemes. CONCLUSION: An SDA of VAR and/or MMR vaccines could be sufficient to protect almost two-thirds of patients. Testing immunogenicity after a single dose of VZ and/or MMR could be included in routine clinical practice to achieve rapid immunization.

A novel live attenuated duck Tembusu virus vaccine targeting N7 methyltransferase protects ducklings against pathogenic strains.

Duck Tembusu virus (DTMUV), an emerging pathogenic flavivirus, causes markedly decreased egg production in laying duck and neurological dysfunction and death in ducklings. Vaccination is currently the most effective means for prevention and control of DTMUV. In previous study, we have found that methyltransferase (MTase) defective DTMUV is attenuated and induces a higher innate immunity. However, it is not clear whether MTase-deficient DTMUV can be used as a live attenuated vaccine (LAV). In this study, we investigated the immunogenicity and immunoprotection of N7-MTase defective recombinant DTMUV K61A, K182A and E218A in ducklings. These three mutants were highly attenuated in both virulence and proliferation in ducklings but still immunogenic. Furthermore, a single-dose immunization with K61A, K182A or E218A could induce robust T cell responses and humoral immune responses, which could protect ducks from the challenge of a lethal-dose of DTMUV-CQW1. Together, this study provides an ideal strategy to design LAVs for DTMUV by targeting N7-MTase without changing the antigen composition. This attenuated strategy targeting N7-MTase may apply to other flaviviruses.