FAdV-4 without Fiber-2 Is a Highly Attenuated and Protective Vaccine Candidate.

Hepatitis-hydropericardium syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) has resulted in huge economic losses to the poultry industry globally. The fiber-2 gene, as a major virulence determiner, is also an important vaccine target against FAdV-4. In this study, we used a CRISPR/Cas9-based homology-dependent recombinant technique to replace the fiber-2 gene with egfp and generate a novel recombinant virus, designated FAdV4-EGFP-rF2. Although FAdV4-EGFP-rF2 showed low replication ability compared to the wild-type FAdV-4 in LMH cells, FAdV4-EGFP-rF2 could effectively replicate in LMH-F2 cells with the expression of Fiber-2. Moreover, FAdV4-EGFP-rF2 was not only highly attenuated in chickens, but also could provide efficient protection against a lethal challenge of FAdV-4. Moreover, FAdV4-EGFP-rF2 without fiber-2 could induce neutralizing antibodies at the same level as FA4-EGFP with fiber-2. These results clearly demonstrate that although fiber-2 affects the viral replication and pathogenesis of FAdV-4, it is not necessary for virus replication and induction of neutralizing antibodies; these findings provide novel insights into the roles of fiber-2 and highlight fiber-2 as an insertion site for generating live-attenuated FAdV-4 vaccines against FAdV-4 and other pathogens. IMPORTANCE Among all serotypes of fowl adenovirus, serotypes FAdV-1, FAdV-4, and FAdV-10 are unique members with two fiber genes (fiber-1 and fiber-2). Recent studies reveal that Fiber-1, not Fiber-2, directly triggers viral infection of FAdV-4, whereas Fiber-2, but not Fiber-1, has been identified as the major virulence determiner and an efficient protective immunogen for subunit vaccines. Here, we replaced fiber-2 with egfp to generate a novel recombinant virus, designated FAdV4-EGFP-rF2. In vitro and in vivo studies on FAdV4-EGFP-rF2 revealed that fiber-2 was not necessary for either virus replication or efficient protection for FAdV-4; these results not only provide a novel live-attenuated vaccine candidate against HHS, but also give new ideas for generating a FAdV-4 based vaccine vector against other pathogens.

Induction of Hepatitis E Virus Anti-ORF3 Antibodies from Systemic Administration of a Muscle-Specific Adeno-Associated Virus (AAV) Vector.

The hepatitis E virus (HEV) is a major global health problem, leading to large outbreaks in the developing world and chronic infections in the developed world. HEV is a non-enveloped virus, which circulates in the blood in a quasi-enveloped form. The quasi-envelope protects HEV particles from neutralising anti-capsid antibodies in the serum; however, most vaccine approaches are designed to induce an immune response against the HEV capsid. In this study, we explored systemic in vivo administration of a novel synthetic and myotropic Adeno-associated virus vector (AAVMYO3) to express the small HEV phosphoprotein ORF3 (found on quasi-enveloped HEV) in the musculature of mice, resulting in the robust and dose-dependent formation of anti-ORF3 antibodies. Neutralisation assays using the serum of ORF3 AAV-transduced mice showed a modest inhibitory effect on the infection of quasi-enveloped HEV in vivo, comparable to previously characterised anti-ORF3 antibodies used as a control. The novel AAVMYO3 capsid used in this study can serve as a versatile platform for the continued development of vector-based vaccines against HEV and other infectious agents, which could complement traditional vaccines akin to the current positive experience with SARS-CoV-2.

Extracellular vesicle-mediated delivery of circDYM alleviates CUS-induced depressive-like behaviours.

Major depressive disorder (MDD) is the most prevalent psychiatric disorder worldwide and severely limits psychosocial function and quality of life, but no effective medication is currently available. Circular RNAs (circRNAs) have been revealed to participate in the MDD pathological process. Targeted delivery of circRNAs without blood-brain barrier (BBB) restriction for remission of MDD represents a promising approach for antidepressant therapy. In this study, RVG-circDYM-extracellular vesicles (RVG-circDYM-EVs) were engineered to target and preferentially transfer circDYM to the brain, and the effect on the pathological process in a chronic unpredictable stress (CUS) mouse model of depression was investigated. The results showed that RVG-circDYM-EVs were successfully purified by ultracentrifugation from overexpressed circDYM HEK 293T cells, and the characterization of RVG-circDYM-EVs was successfully demonstrated in terms of size, morphology and specific markers. Beyond demonstrating proof-of-concept for an RNA drug delivery technology, we observed that systemic administration of RVG-circDYM-EVs efficiently delivered circDYM to the brain, and alleviated CUS-induced depressive-like behaviours, and we discovered that RVG-circDYM-EVs notably inhibited microglial activation, BBB leakiness and peripheral immune cells infiltration, and attenuated astrocyte disfunction induced by CUS. CircDYM can bind mechanistically to the transcription factor TAF1 (TATA-box binding protein associated factor 1), resulting in the decreased expression of its downstream target genes with consequently suppressed neuroinflammation. Taken together, our findings suggest that extracellular vesicle-mediated delivery of circDYM is effective for MDD treatment and promising for clinical applications.

Influenza Neuraminidase Characteristics and Potential as a Vaccine Target.

Neuraminidase of influenza A and B viruses plays a critical role in the virus life cycle and is an important target of the host immune system. Here, we highlight the current understanding of influenza neuraminidase structure, function, antigenicity, immunogenicity, and immune protective potential. Neuraminidase inhibiting antibodies have been recognized as correlates of protection against disease caused by natural or experimental influenza A virus infection in humans. In the past years, we have witnessed an increasing interest in the use of influenza neuraminidase to improve the protective potential of currently used influenza vaccines. A number of well-characterized influenza neuraminidase-specific monoclonal antibodies have been described recently, most of which can protect in experimental challenge models by inhibiting the neuraminidase activity or by Fc receptor-dependent mechanisms. The relative instability of the neuraminidase poses a challenge for protein-based antigen design. We critically review the different solutions that have been proposed to solve this problem, ranging from the inclusion of stabilizing heterologous tetramerizing zippers to the introduction of inter-protomer stabilizing mutations. Computationally engineered neuraminidase antigens have been generated that offer broad, within subtype protection in animal challenge models. We also provide an overview of modern vaccine technology platforms that are compatible with the induction of robust neuraminidase-specific immune responses. In the near future, we will likely see the implementation of influenza vaccines that confront the influenza virus with a double punch: targeting both the hemagglutinin and the neuraminidase.

A Novel Recombinant Influenza Virus Neuraminidase Vaccine Candidate Stabilized by a Measles Virus Phosphoprotein Tetramerization Domain Provides Robust Protection from Virus Challenge in the Mouse Model.

Current seasonal influenza virus vaccines do not induce robust immune responses to neuraminidase. Several factors, including immunodominance of hemagglutinin over neuraminidase, instability of neuraminidase in vaccine formulations, and variable, nonstandardized amounts of neuraminidase in the vaccines, may contribute to this effect. However, vaccines that induce strong antineuraminidase immune responses would be beneficial, as they are highly protective. Furthermore, antigenic drift is slower for neuraminidase than for hemagglutinin, potentially providing broader coverage. Here, we designed stabilized recombinant versions of neuraminidase by replacing the N-terminal cytoplasmic domain, transmembrane, and extracellular stalk with tetramerization domains from the measles or Sendai virus phosphoprotein or from an Arabidopsis thaliana transcription factor. The measles virus tetramerization domain-based construct, termed N1-MPP, was chosen for further evaluation, as it retained antigenicity, neuraminidase activity, and structural integrity and provided robust protection in vivo against lethal virus challenge in the mouse model. We tested N1-MPP as a standalone vaccine, admixed with seasonal influenza virus vaccines, or given with seasonal influenza virus vaccines but in the other leg of the mouse. Admixture with different formulations of seasonal vaccines led to a weak neuraminidase response, suggesting a dominant effect of hemagglutinin over neuraminidase when administered in the same formulation. However, administration of neuraminidase alone or with seasonal vaccine administered in the alternate leg of the mouse induced robust antibody responses. Thus, this recombinant neuraminidase construct is a promising vaccine antigen that may enhance and broaden protection against seasonal influenza viruses. IMPORTANCE Influenza virus infections remain a high risk to human health, causing up to 650,000 deaths worldwide every year, with an enormous burden on the health care system. Since currently available seasonal vaccines are only partially effective and often mismatched to the circulating strains, a broader protective influenza virus vaccine is needed. Here, we generated a recombinant influenza virus vaccine candidate based on the more conserved neuraminidase surface glycoprotein in order to induce a robust and broader protective immune response against a variety of circulating influenza virus strains.

Antigenicity and immunogenicity of recombinant proteins comprising African swine fever virus proteins p30 and p54 fused to a cell-penetrating peptide.

African swine fever (ASF) is a highly fatal swine disease threatening the global pig industry. Currently, vaccine is not commercially available for ASF. Hence, it is desirable to develop effective subunit vaccines against ASF. Here, we expressed and purified two recombinant fusion proteins comprising ASFV proteins p30 and p54 fused to a novel cell-penetrating peptide Z12, which were labeled as ZPM (Z12-p30-modified p54) and ZPMT (Z12-p30-modified p54-T cell epitope). Purified recombinant p30 and modified p54 expressed alone or fused served as controls. The transduction capacity of these recombinant proteins was assessed in RAW264.7 cells. Both ZPM and ZPMT exhibited higher transduction efficiency than the other proteins. Subsequently, humoral and cellular immune responses elicited by these proteins were evaluated in mice. ZPMT elicited the highest levels of antigen-specific IgG responses, cytokines (interleukin-2, interferon-γ, and tumor necrosis factor-α) and lymphocyte proliferation. Importantly, sera from mice immunized with ZPM or ZPMT neutralized greater than 85% of ASFV in vitro. Our results indicate that ZPMT induces potent neutralizing antibody responses and cellular immunity in mice. Therefore, ZPMT may be a suitable candidate to elicit immune responses in swine, providing valuable information for the development of subunit vaccines against ASF.

Immunogenicity and Antigenicity of Rabbit Hepatitis E Virus-Like Particles Produced by Recombinant Baculoviruses.

Rabbit hepatitis E virus (HEV) is a novel HEV belonging to genotype 3 (HEV-3) in the Orthohepevirus A species of the genus Hepevirus, family Hepeviridae. Rabbit HEV was originally isolated from rabbits and found to cause zoonotic infection. Although rabbit HEV can be successfully grown in culture with several cell lines, including the human carcinoma cell line PLC/PRF/5, it is difficult to obtain the large amounts of viral antigen required for diagnosis and vaccine development. In this study, we expressed N-terminal 13 and 111 aa-truncated rabbit HEV ORF2 proteins using recombinant baculoviruses and obtained two types of virus-like particles (VLPs), RnVLPs and RsVLPs with ~35 and 24 nm diameter, respectively. Anti-rabbit HEV IgG antibodies were induced in high titer by immunizing rabbits with RnVLPs or RsVLPs. The antibody secretion in the serum persisted more than three years. RsVLPs showed stronger antigenic cross-reactivity against HEV-1, HEV-3 and HEV-4 than rat HEV. Moreover, anti-RsVLPs antibodies neutralized not only the cognate virus but also HEV-1, HEV-3 and HEV-4 ex vivo, indicating that rabbit HEV had the same serotype as human HEVs. In contrast, the antibody did not block rat HEV infection, demonstrating that rat HEV belonged to a different serotype. Animal experiments indicated that immunization with either RnVLPs or RsVLPs completely protected the rabbits from challenge by rabbit HEV, suggesting that the VLPs are candidates for rabbit HEV vaccine development.

Development of a monoclonal antibody against PRRSV glycoprotein 3 using an immuodominant peptide as immunogen.

Glycoprotein 3 (GP3), a highly glycosylated membrane protein, is a protective antigen and minor structural protein of porcine reproductive and respiratory syndrome virus (PRRSV), and plays a crucial role in virus assembly and infection. In the present study, we synthesized 23 overlapping peptides span GP3 protein sequence and used pig anti-PRRSV serums to identify immunodominant peptides by indirect ELISA. Five immunodominant peptides GP3-P3, P4, P5, P6 and P7 were identified and GP3-P4 (P55LCPTRQAAAEILEPGKS72) was conjugated to carrier protein BSA. One mAb 1E5 against GP3 was generated from BALB/c mice immunized with the conjugates BSA-P4. The Characterization of mAb was identified by Western blot, Dot-ELISA, IPMA and IFA. We found that mAb 1E5 can specifically react with HP-PRRSV strains but not C-PRRSV or NADC30-like PRRSV strains tested in this study. Site-directed alanine substitution analysis revealed that 8 amino acid residues were involved in antibody binding, among them E65, L67 and P69 were critical residue recognized by mAb 1E5. Taken together, this study provided a novel strategy for generating specific mAbs against virus proteins by using immunodominant peptides as targets, and the mAb 1E5 may be useful for development of rapid differential detection method differentiating HP-PRRSV from C-PRRSV and NADC30-like PRRSV.

Genetically engineered exosomes display RVG peptide and selectively enrich a neprilysin variant: a potential formulation for the treatment of Alzheimer's disease.

Exosome is a promising next generation nano-based drug delivery vehicle. However, the unknown molecular mechanisms underlying its natural tissue tropism and the relatively low quantity of naturally enriched molecules of therapeutic value hamper exosome's clinical application. The aim of the research was to create a targeted and highly efficacious exosome formulation for the treatment of Alzheimer's disease (AD). Genetic engineering techniques combined with co-transfection of parental cells were employed to create an exosome formulation that displays RVG peptide on its surface targeting α7-nAChR and simultaneously enriches a neprilysin variant with increased specificity and efficacy in degrading ß amyloid peptide (Aß). The exosome formulation was preferentially internalised into cell lines in an α7-nAChR expression level-dependent manner. When incubated with Aß-producing N2a cells, it significantly decreased intracellular and secreted Aß40 levels, a potency that is superior to exosomes derived from adipose-derived stem cell. When systemically administered into mice, the exosome formulation was preferentially targeted to the hippocampus region of the brain and significantly decreased the expression of proinflammatory genes, IL1α, TNFα and NF-κB, and simultaneously increased the expression of anti-inflammatory gene, IL10. Our exosome formulation may be explored as an over-the-counter treatment for AD.

Single-component multilayered self-assembling nanoparticles presenting rationally designed glycoprotein trimers as Ebola virus vaccines.

Ebola virus (EBOV) glycoprotein (GP) can be recognized by neutralizing antibodies (NAbs) and is the main target for vaccine design. Here, we first investigate the contribution of the stalk and heptad repeat 1-C (HR1C) regions to GP metastability. Specific stalk and HR1C modifications in a mucin-deleted form (GPΔmuc) increase trimer yield, whereas alterations of HR1C exert a more complex effect on thermostability. Crystal structures are determined to validate two rationally designed GPΔmuc trimers in their unliganded state. We then display a modified GPΔmuc trimer on reengineered protein nanoparticles that encapsulate a layer of locking domains (LD) and a cluster of helper T-cell epitopes. In mice and rabbits, GP trimers and nanoparticles elicit cross-ebolavirus NAbs, as well as non-NAbs that enhance pseudovirus infection. Repertoire sequencing reveals quantitative profiles of vaccine-induced B-cell responses. This study demonstrates a promising vaccine strategy for filoviruses, such as EBOV, based on GP stabilization and nanoparticle display.

Characterization of Gene Therapy Associated Uveitis Following Intravitreal Adeno-Associated Virus Injection in Mice.

Purpose: To characterize the intraocular immune cell infiltrate induced by intravitreal adeno-associated virus (AAV) gene therapy. Methods: AAV vectors carrying plasmids expressing green fluorescent protein under the control of PR2.1 were injected intravitreally into AAV naive and AAV primed C57Bl/6 mice. Clinical inflammation was assessed using optical coherence tomography. Intraocular immune cell populations were identified and quantified by flow cytometry on days 1, 7, and 29 after intravitreal injection and compared with sham and fellow eye controls. Results: Optical coherence tomography inflammation score and total CD45+ cell number were significantly higher in AAV injected eyes compared to uninjected fellow eye and sham injected controls. Clinically apparent inflammation (vitritis on optical coherence tomography) and cellular inflammation (CD45+ cell number) was significantly increased in AAV injected eyes and peaked around day 7. Vitritis resolved by day 29, but cellular inflammation persisted through day 29. On day 1, neutrophils and activated monocytes were the dominant cell populations in all AAV injected eyes. On day 7, eyes of AAV exposed animals had significantly more dendritic cells and T cells than eyes of AAV naive animals. By day 29, CD8- T cells were the dominant CD45+ cell population in AAV injected eyes. Conclusions: Intravitreal AAV injection in mice generates clinically evident inflammation that is mild and seems to resolve spontaneously. However, the total number of intraocular CD45+ cells, particularly T cells, remain elevated. Both innate and adaptive immune cells respond to intravitreal AAV regardless of prior immune status, but the adaptive response is delayed in AAV naive eyes.

Engineered Nanoparticle Applications for Recombinant Influenza Vaccines.

Influenza viruses cause seasonal epidemics and represent a pandemic risk. With current vaccine methods struggling to protect populations against emerging strains, there is a demand for a next-generation flu vaccine capable of providing broad protection. Recombinant biotechnology, combined with nanomedicine techniques, could address this demand by increasing immunogenicity and directing immune responses toward conserved antigenic targets on the virus. Various nanoparticle candidates have been tested for use in vaccines, including virus-like particles, protein and carbohydrate nanoconstructs, antigen-carrying lipid particles, and synthetic and inorganic particles modified for antigen presentation. These methods have yielded some promising results, including protection in animal models against antigenically distinct influenza strains, production of antibodies with broad reactivity, and activation of potent T cell responses. Based on the evidence of current research, it is feasible that the next generation of influenza vaccines will combine recombinant antigens with nanoparticle carriers.

The recombinant pseudorabies virus expressing African swine fever virus CD2v protein is safe and effective in mice.

BACKGROUND: African swine fever (ASF) leads to high mortality in domestic pigs and wild boar and is caused by the African swine fever virus (ASFV). Currently, no vaccine is commercially available for prevention, and the epidemic is still spreading. Here, we constructed a recombinant pseudorabies virus (PRV) (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) that expresses the CD2v protein of ASFV and evaluated its effectiveness and safety as a vaccine candidate in mice. METHODS: A homologous recombination fragment containing ASFV CD2v was synthesized and co-transfected into HEK 293 T cells, a knockout vector targeting the PRV TK gene. The transfected cells were infected with PRV-ΔgE/ΔgI, and the recombinant strain (PRV-ΔgE/ΔgI/ΔTK-(CD2v)) was obtained by plaque purification in Vero cells. The expression of ASFV CD2v in the recombinant virus was confirmed by sequencing, Western blotting, and immunofluorescence analysis, and the genetic stability was tested in Vero cells over 20 passages. The virulence, immunogenicity and protective ability of the recombinant virus were further tested in a mouse model. RESULTS: The PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain is stable in Vero cells, and the processing of CD2v does not depend on ASFV infection. The vaccination of PRV-ΔgE/ΔgI/ΔTK-(CD2v) causes neither pruritus, not a systemic infection and inflammation (with the high expression of interleukin-6 (IL6)). Besides, the virus vaccination can produce anti-CD2v specific antibody and activate a specific cellular immune response, and 100% protect mice from the challenge of the virulent strain (PRV-Fa). The detoxification occurs much earlier upon the recombinant virus vaccination and the amount of detoxification is much lower as well. CONCLUSIONS: The PRV-ΔgE/ΔgI/ΔTK-(CD2v) recombinant strain has strong immunogenicity, is safe and effective, and maybe a potential vaccine candidate for the prevention of ASF and Pseudorabies.

Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine.

To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 107 of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (107) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as confirmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4+ T cells and CD4+/CD8+ ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections.

Recombinant Lactobacillus acidophilus expressing S1 and S2 domains of porcine epidemic diarrhea virus could improve the humoral and mucosal immune levels in mice and sows inoculated orally.

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV), which is characterized by a high mortality rate in piglets. Since 2012, a remarkable growth in PED outbreaks occurred in many pig farms in China, landing a heavy blow on the pig industry. In order to develop a new effective vaccine for the current PEDV, oral vaccines were generated by transferring eukaryotic expression recombinant plasmids carrying the S1 and S2 (antigenic sites of the S protein) epitopes of PEDV into a swine-origin Lactobacillus acidophilus (L. acidophilus). After oral immunization of the BALB/c mice, higher levels of anti-PEDV specific IgG and SIgA antibodies and cellular immune responses were detected in mice orally administered with the recombinant L. acidophilus-S1 compared to the L. acidophilus-S2. Furthermore, L. acidophilus-S1 was used to inoculate the pregnant sows orally and the results showed that the recombinant L. acidophilus-S1 could elicit a specific systemic and mucosal immune response. In summary, our study demonstrated that oral immunization with L. acidophilus-S1 could improve the humoral and mucosal immune levels in sows and would be a promising candidate vaccine against PEDV infection in piglets.

A HSV1 mutant leads to an attenuated phenotype and induces immunity with a protective effect.

Herpes simplex virus type 1 (HSV1) is a complicated structural agent with a sophisticated transcription process and a high infection rate. A vaccine against HSV1 is urgently needed. As multiple viral-encoded proteins, including structural and nonstructural proteins, contribute to immune response stimulation, an attenuated or deficient HSV1 vaccine may be relatively reliable. Advances in genomic modification technologies provide reliable means of constructing various HSV vaccine candidates. Based on our previous work, an M6 mutant with mutations in the UL7, UL41, LAT, Us3, Us11 and Us12 genes was established. The mutant exhibited low proliferation in cells and an attenuated phenotype in an animal model. Furthermore, in mice and rhesus monkeys, the mutant can induce remarkable serum neutralizing antibody titers and T cell activation and protect against HSV1 challenge by impeding viral replication, dissemination and pathogenesis.

Two RSV Platforms for G, F, or G+F Proteins VLPs.

Respiratory syncytial virus (RSV) causes substantial lower respiratory tract disease in children and at-risk adults. Though there are no effective anti-viral drugs for acute disease or licensed vaccines for RSV, palivizumab prophylaxis is available for some high risk infants. To support anti-viral and vaccine development efforts, we developed an RSV virus-like particle (VLP) platform to explore the role RSV F and G protein interactions in disease pathogenesis. Since VLPs are immunogenic and a proven platform for licensed human vaccines, we also considered these VLPs as potential vaccine candidates. We developed two RSV VLP platforms, M+P and M+M2-1 that had F and G, F and a G peptide, or a truncated F and G on their surface. Immunoblots of sucrose gradient purified particles showed co-expression of M, G, and F with both VLP platforms. Electron microscopy imaging and immunogold labeling confirmed VLP-like structures with surface exposed projections consistent with F and G proteins. In mice, the VLPs induced both anti-F and -G protein antibodies and, on challenge, reduced lung viral titer and inflammation. These data show that these RSV VLP platforms provide a tool to study the structure of F and G and their interactions and flexible platforms to develop VLP vaccines in which all components contribute to RSV-specific immune responses.

Establishment of a Rep' protein antibody detection method to distinguish natural infection with PCV2 from subunit vaccine immunization.

Introduction. PCV2 is a DNA virus that exists widely in pigs and has caused great economic losses to the pig industry worldwide. In the existing commercial PCV2 enzyme-linked immunosorbent assay (ELISA) kits both natural infection with PCV2 and vaccine immunization produce results that are positive for PCV2 Cap antibodies and therefore they cannot diagnose PCV2 infection in immunized pig farms.Aim. To establish a PCV2 non-structural protein antibody detection method that distinguishes between antibodies resulting from natural prior exposure (infection) and those induced by subunit vaccine immunization.Methodology. Based on the non-structural Rep' protein, we established an indirect ELISA (iELISA) using sera from guinea pigs and piglets.Results. The results for iELISA for guinea pig serum showed that animals vaccinated with a whole-virus inactivated PCV2 vaccine had 100 % (10/10) Cap antibody positivity and 100 % (10/10) Rep' antibody positivity. Guinea pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (10/10) Cap antibody positivity, while no (0/10) guinea pigs were Rep' antibody-positive. The combined detection results for the Rep' iELISA and a PCV2 Antibody Test kit (Commercial) showed that pigs vaccinated with a whole-virus inactivated PCV2 vaccine or PCV2 SD/2017 had 100 % (5/5) Cap antibody positivity and 100 % (5/5) Rep' antibody positivity. Pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (5/5) Cap antibody positivity, while no (0/10) pigs were Rep' antibody-positive.Conclusion. This paper describes an effective iELISA method that can distinguish natural infection with PCV2 (Cap and Rep positive) or inoculation with a whole-virus inactivated vaccine (Cap and Rep positive) from subunit vaccine immunization (Cap-positive, Rep-negative). These comparative assays could be very useful in the control of PCV2 in pig herds.

A Double-Blind, Randomized, Placebo-Controlled Trial of Heat-Killed Pediococcus acidilactici K15 for Prevention of Respiratory Tract Infections among Preschool Children.

Although some probiotic bacteria have been reported to prevent infections in children, there are few well-designed double-blind studies. Here we evaluated the effects of a probiotic strain of lactic acid bacteria (LAB), Pediococcus acidilactici K15, on viral respiratory tract infections in preschool children. A four-month, randomized, double-blind, placebo-controlled study was performed in 172 healthy children aged 3 to 6 years. Subjects were administered dextrin alone or dextrin including heat-killed K15 (5 × 1010 bacteria). The number of febrile days was the primary outcome. The number of absent days from preschools and the influenza incidence were secondary outcomes. Secretory IgA (sIgA) concentrations in saliva were measured as an exploratory outcome. The primary and secondary outcomes were not significantly different between both groups. Analyses in children with little intake of fermented foods including LAB showed that the duration of a fever significantly decreased by K15 intake. The salivary sIgA level in the K15 group was maintained significantly higher than it was in the placebo group. The effects of K15 on preventing viral respiratory tract infections were not observed without the restriction of fermented foods intake. However, K15 supported anti-infectious immune systems in children who took less fermented foods and the maintenance of salivary sIgA levels in all subjects.

The Brighton Collaboration standardized template for collection of key information for benefit-risk assessment of protein vaccines.

Several protein vaccine candidates are among the COVID-19 vaccines in development. The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) has prepared a standardized template to describe the key considerations for the benefit-risk assessment of protein vaccines. This will help key stakeholders to assess potential safety issues and understand the benefit-risk of such a vaccine platform. The structured and standardized assessment provided by the template would also help contribute to improved public acceptance and communication of licensed protein vaccines.