Development and Assessment of Two Highly Pathogenic Avian Influenza(HPAI) H5N6 Candidate Vaccine Viruses for Pandemic Preparedness

Objective In China, 24 cases of human infection with highly pathogenic avian influenza(HPAI) H5 N6 virus have been confirmed since the first confirmed case in 2014. Therefore, we developed and assessed two H5 N6 candidate vaccine viruses(CVVs).Methods In accordance with the World Health Organization(WHO) recommendations, we constructed two reassortant viruses using reverse genetics(RG) technology to match the two different epidemic H5 N6 viruses. We performed complete genome sequencing to determine the genetic stability. We assessed the growth ability of the studied viruses in MDCK cells and conducted a hemagglutination inhibition assay to analyze their antigenicity. Pathogenicity attenuation was also evaluated in vitro and in vivo.Results The results showed that no mutations occurred in hemagglutinin or neuraminidase, and both CVVs retained their original antigenicity. The replication capacity of the two CVVs reached a level similar to that of A/Puerto Rico/8/34 in MDCK cells. The two CVVs showed low pathogenicity in vitro and in vivo, which are in line with the WHO requirements for CVVs.Conclusion We obtained two genetically stable CVVs of HPAI H5N6 with high growth characteristics,which may aid in our preparedness for a potential H5N6 pandemic.

A novel live attenuated vaccine candidate protects chickens against subtype B avian metapneumovirus

Avian metapneumovirus(aMPV) is a highly contagious pathogen that causes acute upper respiratory tract diseases in chickens and turkeys, resulting in serious economic losses. Subtype B aMPV has recently become the dominant epidemic strain in China. We developed an attenuated aMPV subtype B strain by serial passaging in Vero cells and evaluated its safety and efficacy as a vaccine candidate. The safety test showed that after the 30th passage, the LN16-A strain was fully attenuated, as clinical signs of infection and histological lesions were absent after inoculation.The LN16-A strain did not revert to a virulent strain after five serial passages in chickens. The genomic sequence of LN16-A differed from that of the parent wild-type LN16(wtLN16) strain and had nine amino acid mutations. In chickens, a single immunization with LN16-A induced robust humoral and cellular immune responses, including the abundant production of neutralizing antibodies, CD4^(+) T lymphocytes, and the Th1(IFN-γ) and Th2(IL-4 and IL-6)cytokines. We also confirmed that LN16-A provided 100% protection against subtype B aMPV and significantly reduced viral shedding and turbinate inflammation. Our findings suggest that the LN16-A strain is a promising live attenuated vaccine candidate that can prevent infection with subtype B aMPV.

A Natural Vaccine Candidate Strain Against Cholera

E1 Tor Vibrio cholerae (EVC) strains may be classifled into two kinds-epidemigenic (EEVC) strains and non-epidemigenic (NEEVC) strains-based on a phage-biotyping system. A large number of EEVC strains have been screened for toxigenic and putative colonization attributes. One such naturally occurring strain (designated IEM 101) has been found which is devoid of genes encoding cholera toxin (CT), accessory cholera enterotoxin (ACE), zonula occludens toxin (ZOT), but possesses RS1 sequences and toxin-coregulated pilus A gene (tcpA) although tcpA is poorly expressed. It expresses type B pili but does not posses type C pili. It is an E1 Tor Ogawa strain and does not cause fluid accumulation in rabbit ileal loop tests. Active immunization of rabbits with strain IEM 101 elicited good protection against challenge with virulent strains of V cholerae O1. Oral administrationcaused no side effects in 15 human volunteers, colonized the gut for four to ten days and elicited good immune responses

Evaluation of the Protective Efficacy of a Fused OmpK/Omp22 Protein Vaccine Candidate against Acinetobacter baumannii Infection in Mice

Acinetobocter baumannfi （A. Baumannii） is an emerging opportunistic pathogen responsible for hospital-acquired infections, and which now constitutes a sufficiently serious threat to public health to necessitate the development of an effective vaccine. In this study, a recombinant fused protein named OmpK/Omp22 and two individual proteins OmpK and Omp22 were obtained using recombinant expression and Ni-affinity purification. Groups of BALB/c mice were immunized with these proteins and challenged with a clinically isolated strain of A. boumonnii. The bacterial load in the blood, pathological changes in the lung tissue and survival rates after challenge were evaluated. Mice immunized with OmpK/Omp22 fused protein provided significantly greater protection against A. boumonnfi challenge than those immunized with either of the two proteins individually. The results provide novel clues for future design of vaccines against A. boumonnii.

SARS-CoV-2: Vaccines in the pandemic era

Coronavirus disease 2019(COVID-19),caused by the severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),has caused millions of infections and deaths worldwide since its emergence in December 2019.As there is little or no natural immunity in the human population or specific anti-COVID-19 drugs,researchers from the government,academia and industry are developing vaccines at an unprecedented speed to halt the pandemic.In this review,the results of animal experiments and clinical trials on several vaccine technical platforms are summarized,and several challenges are also discussed to further promote the development,evaluation and application of vaccines during the challenging situation of the global pandemic.

Vaccine development for leptospirosis:A systematic review

Objective:To assess the efficacy of various types of vaccines developed for leptospirosis.Methods:A comprehensive search was conducted in three databases:PubMed,Scopus,and Cochrane Library.Two authors(YS and MN)selected the articles based on manual screening.The study eligibility criteria are all Leptospira species regardless of any cluster(pathogenic,intermediate and non-pathogenic).This study recorded articles with positive and negative results and showed a comparison among various membrane proteins as vaccine candidates.The studies on the effectiveness of outer membrane protein as vaccine candidates were also included.The articles obtained in the databases were imported into the WPS spreadsheet,and duplicate documents were removed manually.Results:A total of 24 studies were included in the review,which evaluated various types of leptospirosis vaccines.Multiple vaccines were developed and tested;however,the heterogeneity of Leptospira species pose a challenge.As an effective approach,an epitope based vaccine shows quite a promising result.However,sufficient validation,testing and clinical trials are required.Conclusions:Developing an effective vaccine for leptospirosis remains a global health priority.While significant progress has been made in recent years,there is a need for further research to optimize vaccine development and to ensure that vaccines are accessible and effective for high-risk populations.

In the era of rapid mRNA-based vaccines:Why is there no effective hepatitis C virus vaccine yet?

Hepatitis C virus(HCV)is responsible for no less than 71 million people chronically infected and is one of the most frequent indications for liver transplanta-tion worldwide.Despite direct-acting antiviral therapies fuel optimism in controlling HCV infections,there are several obstacles regarding treatment accessibility and reinfection continues to remain a possibility.Indeed,the majority of new HCV infections in developed countries occur in people who inject drugs and are more plausible to get reinfected.To achieve global epidemic control of this virus the development of an effective prophylactic or therapeutic vaccine becomes a must.The coronavirus disease 19(COVID-19)pandemic led to auspicious vaccine development against severe acute respiratory syndrome coronavirus-2(SARSCoV-2)virus,which has renewed interest on fighting HCV epidemic with vaccination.The aim of this review is to highlight the current situation of HCV vaccine candidates designed to prevent and/or to reduce HCV infectious cases and their complications.We will emphasize on some of the crossroads encountered during vaccine development against this insidious virus,together with some key aspects of HCV immunology which have,so far,ham-pered the progress in this area.The main focus will be on nucleic acid-based as well as recombinant viral vector-based vaccine candidates as the most novel vaccine approaches,some of which have been recently and successfully employed for SARS-CoV-2 vaccines.Finally,some ideas will be presented on which methods to explore for the design of live-attenuated vaccines against HCV.

The Efficacy of a Live Attenuated TW I-Type Infectious Bronchitis Virus Vaccine Candidate

Infectious bronchitis(IB) is a highly contagious avian disease caused by infection with infectious bronchitis virus(IBV),which seriously affects the development of the global poultry industry. The distribution of TW I-type IBV in China has increased in recent years, becoming a widespread genotype. We previously isolated a TW I-type IBV strain termed CK/CH/GD/GZ14 in 2014, but its pathogenicity and possibility for vaccine development were not explored. Therefore, this research aimed to develop a live-attenuated virus vaccine based on the CK/CH/GD/GZ14 strain. The wild type IBV CK/CH/GD/GZ14 strain was serially passaged in SPF embryos for 145 generations. The morbidity and mortality rate of wildtype strain in 14 day-old chickens is 100% and 80% respectively, while the morbidity rate in the attenuated strain was 20%in the 95 th and 105 th generations and there was no death. Histopathological observations showed that the pathogenicity of the 95th and 105th generations in chickens was significantly weakened. Further challenge experiments confirmed that the attenuated CK/CH/GD/GZ14 strain in the 95th and 105 th generations could resist CK/CH/GD/GZ14(5th generation)infection and the protection rate was 80%. Tracheal cilia stagnation, virus shedding, and viral load experiments confirmed that the 95 th and 105th generations provide good immune protection in chickens, and the immunogenicity of the 105th generation is better than that of the 95th generation. These data suggest that the attenuated CK/CH/GD/GZ14 strain in the105th generation may be applied as a vaccine candidate against TW I-type IBV.

RR1 and RR2 gene deletion affects the immunogenicity of a live attenuated pseudorabies virus vaccine candidate in natural pig host

As virulence-determining genes, RR1 and RR2 encode the small subunit and large subunit of viral ribonucleotide reductase(RR) in pseudorabies virus which have been extensively studied in mice. However,their role in pigs has not been adequately investigated. In this study, we deleted RR1 and RR2 genes based on a TK/g E/g I triple gene-deleted pseudorabies virus and tested its efficacy in pigs as a vaccine candidate. The rescued virus showed similar growth properties and plaque size in vitro as its parent strain. In an animal study, the virus could elicit humoral immune responses shown by generation of g B-specific antibodies and virus neutralizing antibodies.However, vaccination could not provide protection against virulent pseudorabies virus challenge since vaccinated pigs showed clinical pseudorabies-specific syndromes. The deficiency in protection may due to the generation of late and low levels of gB antibodies and virus neutralizing antibodies.

Neospora caninum immune mapped protein 1(NcIMP1) is a novel vaccine candidate against neosporosis

The Neospora caninum immune mapped protein 1(Nc IMP1) was identified as a membrane protein,and a previous study indicated that Nc IMP1 could be a promising vaccine candidate against neosporosis. In this study, the immune response and protection efficacy of Nc IMP1 were evaluated. The coding sequence of Nc IMP1 was inserted into the eukaryotic expression vector pc DNA3.1(+), resulting in the recombination plasmid pc DNAIMP1, which was used for the intramuscular immunization of BALB/c mice. After immunization, the immune response was evaluated using a lymphoproliferative assay and cytokine and antibody measurements. Quantification of the cerebral parasite burden of mice challenged with 2106 N. caninum was performed 14 days after the last immunization. The results showed that the mice immunized with pc DNA-IMP1 developed a high level of specific antibody responses against recombinant Nc IMP1,with a mixed Ig G1/Ig G2 a response and a predominance of Ig G2 a production. The cellular immune response was associated with the production of IFN-γ, IL-2, IL-4 and IL-10 cytokines. The experiment was terminated 30 days p.i.,and the cerebral parasite burden in each mouse was assessed by quantitative PCR. The parasite burden was significantly reduced in the pc DNA-IMP1-vaccinated mice. These data suggest that IMP1 is a promising vaccine candidate against neosporosis.

Antigenicity of Synthetic Peptides Derived from Plasmodium Apoptosis-Linked Pathogenicity Factors

Background： Plasmodium falciparum malaria remains a major life-threatening disease. Recently, the Plasmodium apoptosis-linked pathogenicity factors （PALPF） have been identified. These antigens PALPF are expressed only by P falciparum-infected erythrocytes triggering endothelial cell apoptosis （apoptogenic）. Methods： We designed ten synthetic peptides （PI to P10） from PALPF： PF07 0032, PF10_0226, PFI0130c, PFD0875c and MAL13P1.206, and analyzed their antigenicity with an ELISA method using plasma samples from subjects living in Dienga, Gabon. Results： Four peptides showed good reactivity with human antibodies. The prevalence rate of specific IgG was 61%, 51%, 44% and 34% for P5, P6, P4 and P2, respectively. The median optical density of total IgG anti-P2 was higher than that directed against P4 and P6 （P = 0.009; P = 0.012 respectively）. The prevalence rate oflgG subclasses determined with plasma samples recognizing peptide 5 for IgGl, 2, 3 and 4 isotypes was 69%, 45%, 76% and 62%, respectively. All the subjects had at least one immunoglobulin subclass, while 13 （44%） had both IgG1 and IgG3 antibodies. There was no significant difference in the prevalence rate of anti-P5 IgG1, IgG3 and IgG4. Conclusion： These results warrant further immunogenicity studies of peptides 2, 4, 5 and 6 with a view of a tentative to antimalarial vaccine development.

A new can didate vaccine for human brucellosis based on influenza viral vectors:a preliminary investigation for the development of an immunization schedule in a guinea pig model

Background:A new candidate vector vaccine against human brucellosis based on recombinant influenza viral vectors(rIVV)subtypes H5N1 expressing Brucella outer membrane protein(Omp)16,L7/L12,Omp19or Cu-Zn SOD proteins has been developed.This paper presents the results of the study of protection of the vaccine using on guinea pigs,including various options of administering,dose and frequency.Provided data of the novel vaccine candidate will contribute to its further movement into the preclinical stage study.Methods:General states of guinea pigs was assessed based on behavior and dynamics of a guinea pig weight-gain test.The effectiveness of the new anti-brucellosis vector vaccine was determined by studying its protective effect after conjunctival,intranasal and sublingual administration in doses 10^(5) EID50,10^(6) EID_(50) and 10^(7) EID_(50) during prime and boost vaccinations of animals,followed by challenge with a virulent strain of B.melitensis 16 M infection.For sake of comparison,the commercial ft melitensis Rev.1 vaccine was used as a control.The protective properties of vaccines were assessed by quantitation of Brucella colonization in organs and tissues of infected animals and compared to the control groups.Results:It was observed a gradual increase in body weight of guinea pigs after prime and booster immunization with the vacci ne using conjunctival,intra nasal and subli ngual routes of administration,as well as after using various doses of vaccine.The most optimal way of using the vaccine has been established:double intranasal immunization of guinea pigs at a dose of 10^(6) EID50, which provides 80%protection of guinea pigs from B.melitensis 16 M infection(P<0.05),which is comparable to the results of the effectiv en ess of the commercial B.melitensis Rev.1 vacci ne.Conclusions:We developed effective human vaccine candidate against brucellosis and developed its immunization protocol in guinea pig model.We believe that because of these studies,the proposed vaccine has achieved the best level of protection,which in turn provides a basis for its further promotion.

Chimeric Newcastle Disease Virus-like Particles Containing DC-Binding Peptide-Fused Haemagglutinin Protect Chickens from Virulent Newcastle Disease Virus and H9N2 Avian Influenza Virus Challenge

Newcastle disease virus(NDV)and H9N2 subtype Avian influenza virus(AIV)are two notorious avian respiratory pathogens that cause great losses in the poultry industry.Current inactivated commercial vaccines against NDV and AIV have the disadvantages of inadequate mucosal responses,while an attenuated live vaccine bears the risk of mutation.Dendritic cell(DC)targeting strategies are attractive for their potent mucosal and adaptive immune-stimulating ability against respiratory pathogens.In this study,DC-binding peptide(DCpep)-decorated chimeric virus-like particles(cVLPs),containing NDV haemagglutinin–neuraminidase(HN)and AIV haemagglutinin(HA),were developed as a DC-targeting mucosal vaccine candidate.DCpep-decorated cVLPs activated DCs in vitro,and induced potent immune stimulation in chickens,with enhanced secretory immunoglobulin A(sIgA)secretion and splenic T cell differentiation.40μg cVLPs can provide full protection against the challenge with homologous,heterologous NDV strains,and AIV H9N2.In addition,DCpep-decorated cVLPs could induce a better immune response when administered intranasally than intramuscularly,as indicated by robust s IgA secretion and a reduced virus shedding period.Taken together,this chimericVLPs are a promising vaccine candidate to control NDV and AIV H9N2 and a useful platform bearing multivalent antigens.