Deyan Luo

Protective Immunity Elicited by a Divalent DNA Vaccine Encoding Both the L7/L12 and Omp16 Genes of Brucella abortus in BALB/c Mice

ABSTRACT This study was designed to evaluate the immunogenicity and the protective efficacy of a divalent fusion DNA vaccine encoding both the Brucella abortus L7/L12 protein (ribosomal protein) and Omp16 protein (outer membrane lipoprotein), designated pcDNA3.1-L7/L12-Omp16. Intramuscular injection of this divalent DNA vaccine into BALB/c mice elicited markedly both humoral and cellular immune responses. The specific antibodies exhibited a dominance of immunoglobulin G2a (IgG2a) over IgG1. In addition, the dual-gene DNA vaccine elicited a strong T-cell proliferative response and induced a large amount of gamma interferon-producing T cells upon restimulation in vitro with recombinant fusion protein L7/L12-Omp16, suggesting the induction of a typical T-helper-1-dominated immune response in vivo. This divalent DNA vaccine could also induce a significant level of protection against challenge with the virulent strain B. abortus 544 in BALB/c mice. Furthermore, the protection level induced by the divalent DNA vaccine was significantly higher than that induced by the univalent DNA vaccines pcDNA3.1-L7/L12 or pcDNA3.1-Omp16. Taken together, the results of this study verify for the first time that the Omp16 gene can be a candidate target for a DNA vaccine against brucellosis. Additionally, a divalent genetic vaccine based on the L7/L12 and Omp16 genes can elicit a stronger cellular immune response and better immunoprotection than the relevant univalent vaccines can.

Protection of mice from Brucella infection by immunization with attenuated Salmonella enterica serovar typhimurium expressing A L7/L12 and BLS fusion antigen of Brucella.

This study describes the potential use of attenuated Salmonella enterica serovar Typhimurium Strains (S. typhimurium) to express and deliver a L7/L12 and BLS fusion antigen of Brucella as a vaccination strategy to prevent Brucella infection in mice. S. typhimurium X4072 that contained a pTrc99A-BLS-L7/L12 plasmid, designated X4072bl, can deliver a L7/L12 and BLS fusion antigen expressed by the bacterium itself, while S. typhimurium X4550 that contained an asd-pVAX1-BLS-L7/L12 (asd-pBL) plasmid, designated X4550bl, can deliver the antigen to be expressed in target eukaryotic cells. When orally administered to BALB/c mice, both attenuated carrier strains were able to elicit mucosal and systemic immunity, which induced protection against B. abortus 544 infection in mice. The immunogenicity and protective efficacy of X4072bl and X4550bl were compared with a recombinant BLS-L7/L12 fusion protein vaccine (rBL) and a pVAX1-BLS-L7/L12 DNA vaccine (pBL) in this study. When rBL and pBL were intramuscularly injected into mice, both vaccines could also elicit comparable humoral and cellular immune responses, but not mucosal immunity, which therefore induced lower protection. Taken together, Salmonella-based subunit vaccines are a promising vaccine strategy in the prevention of Brucella infection.

Immunogenicity and protective efficacy of a live attenuated vaccine against the 2009 pandemic A H1N1 in mice and ferrets.

A novel 2009 influenza A (H1N1) virus was transmitted from humans to humans worldwide. The live attenuated monovalent A H1N1 vaccine (LAMV) for intranasal administration has shown promising immunogenicity and safety in clinical trials and for human use, but the experimental data based on LAMV is incomplete. In this study, using reverse genetic technology, we produced a cold-adapted (ca), live attenuated BJ/AA ca that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2009 pandemic A H1N1 isolate, A/Beijing/501/2009 virus (BJ501), and the remaining six internal gene segments from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). BJ/AA ca exhibited phenotypes of temperature sensitivity (ts), ca, and attenuation (att). The candidate BJ/AA ca was immunogenic in mice and induced strong mucosal secretory IgA (sIgA) in the respiratory tract. Two dosages of intranasal immunization induced robust HI antibodies and offered efficient protection against challenge by the wild-type (wt) 2009 pandemic A H1N1 (A/Beijing/501/2009 or A/California/07/2009) in mice and ferrets. These results support the evaluation of this vaccine made from a wt strain isolated in China for clinical trials.

Identification of immunoreactive proteins of Brucella melitensis by immunoproteomics

Infection with Brucella causes brucellosis, a chronic disease in humans, which induces abortion and sterility in livestock. Among the different Brucella species, Brucella melitensis is considered the most virulent and is the predominant species associated with outbreaks in China. To date, no safe human vaccine is available against Brucella infection. The currently used live vaccines against Brucella in livestock induce antibodies that interfere with the diagnosis of field infection in vaccinated animals, which is harmful to eradication programs. However, there is as yet no complete profile of immunogenic proteins of B. melitensis. Towards the development of a safer, equally efficacious, and field infection-distinguishable vaccine, we used immunoproteomics to identify novel candidate immunogenic proteins from B. melitensis M5. Eighty-eight immunoreactive protein spots from B. melitensis M5 were identified by Western blotting and were assigned to sixty-one proteins by mass spectrometry, including many new immunoreactive proteins such as elongation factor G, F0F1 ATP synthase subunit beta, and OMP1. These provide many candidate immunoreactive proteins for vaccine development.

Cross-clade protection against HPAI H5N1 influenza virus challenge in BALB/c mice intranasally administered adjuvant-combined influenza vaccine

The avian H5N1 influenza virus has the potential to cause a new pandemic. The increasing number of recent outbreaks of highly pathogenic avian influenza H5N1 in birds and humans emphasizes the urgent need to develop a potent H5N1 vaccine. Here, we studied the immunogenicity and protective effect of a vaccine prepared from H5N1 inactivated whole virus. This vaccine was intranasally co-administered in mice with phosphate buffered saline, recombinant cholera toxin B subunit (rCTB), cholera toxin (CT), rCTB containing a trace amount of holotoxin (rCTB/CT), polyinosinic:polycytidylic acid double-stranded RNA (polyI:C), or MF59 as an adjuvant. Intranasal administration of H5N1 inactivated whole virus vaccine with rCTB, CT, rCTB/CT, polyI:C, and MF59 elicited an immunological response with both secretory IgA (sIgA) in nasal, lung, and vaginal lavage, and IgG antibody in serum, showing protective immunity against lethal H5N1 infection. Cross-clade protection was also observed in animals immunized with a vaccine derived from Anhui/01/2005(H5N1) with rCTB, CT, rCTB/CT, polyI:C, or MF59 as adjuvants that were subsequently challenged with the A/OT/SZ/097/03 influenza strain.

Downregulation of angiotensin-converting enzyme 2 by the neuraminidase protein of influenza A (H1N1) virus

Abstract Influenza A (H1N1) virus, a high-risk infectious pathogen, can cause severe acute lung injury leading to significant morbidity and mortality. Angiotensin-converting enzyme 2 (ACE2), a negative regulator of the renin-angiotensin system (RAS), plays a protective role in pathogenesis of acute lung injury. Here, we showed that ACE2 protein levels were significantly downregulated after infection with H1N1 viruses but was dispensable for viral replication. ACE2 protein downregulation was most likely related to ACE2 protein degradation by proteasome pathway rather than ACE2 shedding. Finally, we found that ACE2 cleavage could be regulated by influenza neuraminidase (NA), which was fundamentally different from the classically sheddase-induced proteolytic cleavage of ACE2.

Characterization of a highly pathogenic avian influenza H5N1 virus isolated from an ostrich.

The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among poultry and wild birds has posed a potential threat to human public health. An influenza pandemic happens, when a new subtype that has not previously circulated in humans emerges. Almost all of the influenza pandemics in history have originated from avian influenza viruses (AIV). Birds are significant reservoirs of influenza viruses. In the present study, we performed a survey of avian influenza virus in ostriches and H5N1 virus (A/Ostrich/SuZhou/097/03, China097) was isolated. This H5N1 virus is highly pathogenic to both chickens and mice. It is also able to replicate in the lungs of, and to cause death in, BALB/c mice following intranasal administration. It forms plaques in chicken embryo fibroblast (CEF) cells in the absence of trypsin. The hemagglutinin (HA) gene of the virus is genetically similar to A/Goose/Guangdong/1/96(H5N1) and belongs to clade 0. The HA sequence contains multiple basic amino acids adjacent to the cleavage site, a motif associated with HPAI viruses. More importantly, the existence of H5N1 isolates in ostriches highlights the potential threat of wild bird infections to veterinary and public health.

Cytosolic Low Molecular Weight FGF2 Orchestrates RIG-I–Mediated Innate Immune Response

Fibroblast growth factor (FGF)2,which is one of the 22 members of the FGF family, functions as an extracellular molecule involved in canonical receptor tyrosine kinase signaling. It has been implicated in angiogenesis and the development of the CNS. In this article, we reveal that cytosolic low m.w. isoform (LMW) FGF2 (18 kDa), not its secreted form, plays an unexpected role in the innate immune response. Cytosolic LMW FGF2 directly associated with inactivated RIG-I under physiological conditions, which enhanced RIG-I protein stability, thereby maintaining basal RIG-I levels. However, during RIG-I activation induced by viral RNA, cytosolic FGF2 bound to the caspase recruitment domains of activated RIG-I, which blocked RIG-I–MAVS complex formation. LMW FGF2 deficiency increased type I IFN production, whereas the overexpression of LMW FGF2 exerted the opposite effect. Cytosolic LMW FGF2 functions as a negative regulator in RIG-I–mediated antiviral signaling. This work provides insight into the role of FGF2 in innate immune response.

Rescue and Immunogenic Evaluation of Attenuated Vaccine Candidate From A/California/07/2009: Rescue and Immunogenic Evaluation of Attenuated Vaccine Candidate From A/California/07/2009

Six internal protein gene segments of attenuated,cold-adapted(ca),temperature-sensitive(ts) influenza A/Ann Arbor/6/60 ca(H2N2) and HA,NA gene segments of A/California/07/2009ca were introduced to plasmid pAD3000 carrying polⅠand polⅡpromoters,and rescued the reassortant virus from Vero cell using reverse genetics technology.The reassortant has the attenuate characters,ca and ts,the TCID50 is 7.5,HA titer maintain at 1:256 and EID50 is 8 which was detected using SPF eggs.The stable of reassortant is determined by RT-PCR gene segments from virus which were propagated in eggs.The morphology of reassortant conform the wild type virus.In order to test the immunogenicity,the reassortant viruses were purified.Mice were intranasally immunized and intramuscular injected with inactive whole virus as control.The high HI titer can be detected in both groups,seems hinger in i.m.(intramuscular) immunized groups(P=0.044),but higher IgA titer can only detected in i.n.(intranasal) immunized group.Compared with i.m.immunized group,higer pro-inflammation cytokines IL-1β,TNFα,IFN-α were tested in i.n.groups,means faster and stronger mucosal immune response was induced.Virus load were detected in lung,brain,spleen 4 days after immunization to determine the safety of reassortant as vaccine candidate,no detectable virus were found.All results show that the vaccine candidate can be used for vaccine production.

Development and Identification of a Live Attenuated Influenza B Virus Vaccine Candidate

A cold-adapted (ca), temperature sensitive (ts), live attenuated influenza B virus strain B/Ann Arbor/1/66 was chosen influenza virus rescue research, in which six internal gene segments, PB 1, PB2, PA, NP, M, NS, were fully synthesized and nine amino acid substitutions were artificially alter by human intervention. The resultant B/Ann Arbor/1/66 plasmids were named as pAB121-PBI, pAB 122-PB2, pAB 123-PA, pAB 124-HA, pAB 125-NP, pAB126-NA, pAB 127-M and pAB 128-NS, respectively. A recombinant influenza A virus was previously generated entirely from cloned eDNA. An infectious recombinant influenza B virus was generated here, and designated as rMDV-B, by plasmid-based reverse genetics. The rMDV-B virus contained HA and NA genes from epidemic influenza B virus strain B/MalaysiaJ2506/2004 in the background of internal genes derived from influenza B vims strain B/Aim Arbor/1/66. HA titer of rMDV-B in MDCK cells and embryonated chicken eggs ranged from 1 64 to 1 512. The results may allow an effective live influenza B vaccine to be produced from a single master strain, providing a model for the design of future human influenza vaccines.