<i>Salmonella</i> Gallinarum delivering M2eCD40L in protein and DNA formats acts as a bivalent vaccine against fowl typhoid and H9N2 infection in chickens

Hajam, Irshad Ahmed,Kim, Jehyoung,Lee, John Hwa BioMed Central 2018 Veterinary research Vol.49 No.-

<P>Fowl typhoid (FT), a septicemic disease caused by <I>Salmonella</I> Gallinarum (SG), and H9N2 influenza infection are two economically important diseases that affect poultry industry worldwide. Herein, we exploited a live attenuated SG mutant (JOL967) to deliver highly conserved extracellular domains of H9N2 M2 (M2e) to induce protective immunity against both H9N2 infection and FT. To increase the immunogenicity of M2e, we physically linked it with CD40L and cloned the fusion gene into either prokaryotic constitutive expression vector pJHL65 or mammalian expression vector pcDNA3.1+. Then pJHL65-M2eCD40L or pcDNA-M2eCD40L recombinant plasmid was electroporated into JOL967 strain and the resultant clones were designated as JOL2074 and JOL2076, respectively. We demonstrated that the chickens vaccinated once orally with a co-mix of JOL2074 and JOL2076 strains elicited significantly (<I>p</I> < 0.05) higher M2e-specific humoral and cell-mediated immunity compared to JOL2074 alone vaccinated group. However, SG-specific immune responses were comparable in both the vaccination groups. On challenge with the virulent H9N2 virus (10<SUP>5 </SUP>TCID<SUB>50</SUB>) at 28<SUP>th</SUP> day post-vaccination, chickens that received a co-mix of JOL2074 plus JOL2076 strains exhibited significantly (<I>p</I> < 0.05) lower lung inflammation and viral load in both lungs and cloacal samples than JOL2074 alone vaccinated group. Against challenge with the lethal wild-type SG, both the vaccination groups exhibited only 12.5% mortality compared to 75% mortality observed in the control group. In conclusion, we show that SG delivering M2eCD40L can act as a bivalent vaccine against FT and H9N2 infection and further studies are warranted to develop this SG-M2eCD40L vaccine as a broadly protective vaccine against avian influenza virus subtypes.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1186/s13567-018-0593-z) contains supplementary material, which is available to authorized users.</P>

Incorporation of membrane-anchored flagellin into <i>Salmonella</i> Gallinarum bacterial ghosts induces early immune responses and protection against fowl typhoid in young layer chickens

Hajam, Irshad Ahmed,Kim, Je Hyoung,Lee, John Hwa Elsevier 2018 Veterinary immunology and immunopathology Vol.199 No.-

<P><B>Abstract</B></P> <P>The present study aimed to investigate whether the incorporation of flagellin, a TLR5 agonist, in the bacterial ghosts (BGs) of <I>Salmonella</I> Gallinarum can enhance protective immune responses against fowl typhoid, a septicemic disease of poultry, in chickens. BGs are empty cell envelopes derived from Gram-negative bacteria through the bacteriophage phiX174 gene <I>E</I> mediated lysis. In this study, the <I>S.</I> Gallinarum ghosts carrying flagellin were genetically constructed utilizing a lysis plasmid pJHL184-flagellin, designed for the coexpression of the flagellin and the lysis protein E. The adjuvant effect of flagellin was evaluated by immunizing seven day old brown nick layer chicks once orally with either <I>S.</I> Gallinarum-flagellin (SG-fliC) ghosts or <I>S.</I> Gallinarum (SG) ghosts alone. Our results showed that immunization with the SG-fliC ghosts elicited early and higher systemic (IgG) and mucosal (IgA) antibody responses compared to the SG ghosts alone, although not always statistically significant. Flow cytometric analysis of the CD3 + CD4+ and the CD3 + CD8+ T cell populations in peripheral blood mononuclear cells were higher in chickens immunized with the SG-fliC ghosts compared to the chickens vaccinated with the SG ghosts alone. Furthermore, the chickens immunized with SG-fliC ghosts exhibited significantly (p < 0.05) higher IL-6 and IFN-<B>γ</B> responses compared to the chickens vaccinated with the SG ghosts alone. On challenge with the virulent <I>S</I>. Gallinarum wild type strain at 28th day post immunization, 5 of 10 birds died (50%) in case of SG-fliC ghost group while 60% (6 of 10 birds died) mortality was observed in the SG ghost group. Collectively, these results suggest that the expression of flagellin in SG ghosts improves antigen-specific humoral and cell mediated immune responses, and can enhance protective efficacy of the BG-based vaccines against the virulent challenges.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Flagellin (fliC) in Salmonella Gallinarium (SG) ghosts retain TLR5 binding activity. </LI> <LI> SG-fliC ghosts were completely safe in seven day old layer chicks. </LI> <LI> SG-fliC ghosts mediated early induction of systemic and mucosal antibody responses. </LI> <LI> fliC in SG ghosts enhanced cell mediated immunity and immune protection against fowl typhoid in chickens. </LI> </UL> </P>

Oral immunization with a novel attenuated Salmonella Gallinarum encoding infectious bronchitis virus spike protein induces protective immune responses against fowl typhoid and infectious bronchitis in chickens

Hajam, Irshad Ahmed,Kim, Jehyoung,Lee, John Hwa Springer (Biomed Central Ltd.) 2018 Veterinary research Vol.49 No.-

Bacterial flagellin—a potent immunomodulatory agent

HAJAM IRSHAD AHMED,Pervaiz A Dar,Imam Shahnawaz,Juan Carlos Jaume,이존화 생화학분자생물학회 2017 Experimental and molecular medicine Vol.49 No.-

Flagellin is a subunit protein of the flagellum, a whip-like appendage that enables bacterial motility. Traditionally, flagellin was viewed as a virulence factor that contributes to the adhesion and invasion of host cells, but now it has emerged as a potent immune activator, shaping both the innate and adaptive arms of immunity during microbial infections. In this review, we summarize our understanding of bacterial flagellin and host immune system interactions and the role flagellin as an adjuvant, anti-tumor and radioprotective agent, and we address important areas of future research interests.

Immunogenicity of a bacterial ghost delivered dengue virus serotype 2 envelope protein domain III followed by an intramuscular protein boosting strategy in mice model

Gayeon Won,Irshad Ahmed Hajam,Perumalraja Kirthika,Eunha Kim,John Hwa Lee 한국예방수의학회 2022 예방수의학회지 Vol.46 No.3

This study aimed to investigate whether bacterial ghosts (BGs), empty cell envelopes of a gram-negative bacterium, delivering envelope protein domain III (EDIII) of dengue virus (DENV) serotype 2 could induce protective immune responses against dengue infection. In this study, we constructed Salmonella Typhimurium BGs expressing and delivering EDIII (BG-EDIII) and evaluated these ghosts for their immunogenicity studies in C57BL/6 mice. Our results demonstrated that the mice vaccinated once orally with BG-EDIII followed by an intramuscular boosting with a recombinant EDIII protein elicited significantly higher humoral and cell-mediated immune responses compared to the BGs alone vaccinated group (p<0.001). Upon challenge with DENV2, significantly lower viral load and liver damage was observed in BG-EDIII vaccinated group than BGs alone control group (p<0.05). The outcomes of this study revealed the ability of BG- EDIII to stimulate immune response with no observable damage to the vital organs.

Oral immunization with a novel attenuated <i>Salmonella</i> Typhimurium encoding influenza HA, M2e and NA antigens protects chickens against H7N9 infection

Kim, Je Hyoung,Hajam, Irshad Ahmed,Lee, John Hwa BioMed Central 2018 VETERINARY RESEARCH Vol.49 No.-

<P>Attenuated <I>Salmonella</I> strains constitute a promising technology for the development of efficient protein-based influenza vaccines. H7N9, a low pathogenic avian influenza (LPAI) virus, is a major public health concern and currently there are no effective vaccines against this subtype. Herein, we constructed a novel attenuated <I>Salmonella</I> Typhimurium strain for the delivery and expression of H7N9 hemagglutinin (HA), neuraminidase (NA) or the conserved extracellular domain of the matrix protein 2 (M2e). We demonstrated that the constructed <I>Salmonella</I> strains exhibited efficient HA, NA and M2e expressions, respectively, and the constructs were safe and immunogenic in chickens. Our results showed that chickens immunized once orally with <I>Salmonella</I> (Sal) mutants encoding HA (Sal-HA), M2e (Sal-M2e) or NA (Sal-NA), administered either alone or in combination, induced both antigen-specific humoral and cell mediated immune (CMI) responses, and protected chickens against the lethal H7N9 challenge. However, chickens immunized with Sal-HA+Sal-M2e+Sal-NA vaccine constructs exhibited efficient mucosal and CMI responses compared to the chickens that received only Sal-HA, Sal-M2e or Sal-M2e+Sal-NA vaccine. Further, chickens immunized with Sal-HA+Sal-M2e+Sal-NA constructs cleared H7N9 infection at a faster rate compared to the chickens that were vaccinated with Sal-HA, Sal-M2e or Sal-M2e+Sal-NA, as indicated by the reduced viral shedding in cloacal swabs of the immunized chickens. We conclude that this vaccination strategy, based on HA, M2e and NA, stimulated efficient induction of immune protection against the lethal H7N9 LPAI virus and, therefore, further studies are warranted to develop this approach as a potential prophylaxis against LPAI viruses affecting poultry birds.</P><P><B>Electronic supplementary material</B></P><P>The online version of this article (10.1186/s13567-018-0509-y) contains supplementary material, which is available to authorized users.</P>

Orally administered live attenuated <i>Salmonella</i> Typhimurium protects mice against lethal infection with H1N1 influenza virus

Kamble, Nitin Machindra,Hajam, Irshad Ahmed,Lee, John Hwa Elsevier Scientific Pub. Co 2017 Veterinary microbiology Vol.201 No.-

<P><B>Abstract</B></P> <P>Pre-stimulation of toll-like receptors (TLRs) by agonists has been shown to increase protection against influenza virus infection. In this study, we evaluated the protective response generated against influenza A/Puerto Rico/8/1934 (PR8; H1N1) virus by oral and nasal administration of live attenuated <I>Salmonella enterica</I> serovar Typhimurium, JOL911 strain, in mice. Oral and nasal inoculation of JOL911 significantly increased the mRNA copy number of TLR-2, TLR4 and TLR5, and downstream type I interferon (IFN) molecules, IFN-α and IFN-β, both in peripheral blood mononuclear cells (PBMCs) and in lung tissue. Similarly, the mRNA copy number of interferon-inducible genes (ISGs), Mx and ISG15, were significantly increased in both the orally and the nasally inoculated mice. Post PR8 virus lethal challenge, the nasal JOL911 and the PBS control group mice showed significant loss of body weight with 70% and 100% mortality, respectively, compared to only 30% mortality in the oral JOL911 group mice. Post sub-lethal challenge, the significant reduction in PR8 virus copy number in lung tissue was observed in oral [on day 4 and 6 post-challenge (dpc)] and nasal (on 4dpc) than the PBS control group mice. The lethal and sub-lethal challenge showed that the generated stimulated innate resistance (StIR) in JOL911 inoculated mice conferred resistance to acute and initial influenza infection but might not be sufficient to prevent the PR8 virus invasion and replication in the lung. Overall, the present study indicates that oral administration of attenuated <I>S.</I> Typhimurium can pre-stimulate multiple TLR pathways in mice to provide immediate early StIR against a lethal H1N1 virus challenge.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Pre-stimulation of TLR pathways provides stimulated innate resistance (StIR) against influenza virus. </LI> <LI> <I>S.</I> Typhimurium activates multiple TLR pathways, downstream interferons and effector interferon stimulated genes in PBMCs and in lung tissues. </LI> <LI> Oral administration of attenuated <I>S.</I> Typhimurium elicit StIR against PR8 influenza virus. </LI> <LI> The StIR generated in response to <I>S.</I> Typhimurium can protect mice against lethal challenge of PR8 influenza virus. </LI> </UL> </P>