Shigella virulence protein VirG is a broadly protective antigen and vaccine candidate.

Diarrhea caused by Shigella has been associated with high morbidity and mortality in young children worldwide. There are no licensed vaccines, and those clinically advanced have restricted coverage as they elicit serotype-specific immunity while disease is caused by multiple circulating serotypes. Our group had previously reported a close association between serum antibodies to the Shigella virulence factor VirG (or IcsA) and clinical protection in infected individuals. VirG is highly conserved among Shigella strains and appealing as a broad-spectrum vaccine candidate. In this study, we investigated the immunogenicity and protective capacity of VirG as a subunit vaccine in mice. The surface-exposed alpha (α) domain of VirG (VirGα) was produced as a recombinant protein. This region has almost identical immune reactivity to full-length VirG. Administered intramuscularly with alum, VirGα elicited robust immune responses and high protective efficacy against S. flexneri 2a and S. sonnei. Almost complete protection was afforded by VirGα given intranasally with the E. coli double mutant heat-labile toxin (dmLT). VirGα-specific antibodies recognized VirG expressed on live Shigella, and blocked Shigella adhesion and invasion to human colonic cells. These results show for the first time that VirGα is a promising cross-protective vaccine candidate to prevent Shigella infection.

Molecular typing and pathogenicity assessment of fowl adenovirus associated with inclusion body hepatitis in chicken from India.

Recently, inclusion body hepatitis (IBH) outbreaks have been increasingly reported in different regions of India, particularly in broiler flocks. The present study was undertaken to characterize fowl adenovirus associated with IBH in chicken and assessment of its pathogenicity. Liver samples were collected from fowl adenovirus (FAdV) suspected 100 commercial broiler and six broiler breeder flocks from eleven different States of India from 2016 to 2019. All the samples were subjected to 897-bp FAdV hexon gene-specific PCR for confirmation and primary chicken liver cells were used to isolate the field FAdVs. Sequencing and phylogenetic analysis of 897-bp FAdV hexon gene revealed that all the isolates have showed close evolutionary relationship with fowl adenovirus serotype 11 of species D. For pathogenicity assessment, 0.5 ml of 106.5 TCID50/ml of field FAdV serotype 11 isolate was orally inoculated in 1-day-old SPF chicks and observed for 21 days. This experimental study revealed that there was no mortality in infected chicks and showed clinical signs of dullness, depression and diarrhoea between third and fifth day of oral inoculation. The FAdV was reisolated and confirmed by PCR from experimentally infected chicken. Based on this study, among all serotypes, FAdV serotype 11 is involved in pathogenesis of inclusion body hepatitis in broiler-type chickens in India.

Development, Interlaboratory Evaluations, and Application of a Simple, High-Throughput Shigella Serum Bactericidal Assay.

Shigella is an important cause of diarrhea worldwide, with serotypes Shigella flexneri 2a, S. flexneri 3a, and Shigella sonnei demonstrating epidemiological prevalence. Many development efforts are focused on Shigella lipopolysaccharide (LPS)-based vaccines, as O antigen-specific conjugate vaccines are immunogenic and efficacious. Immunization with Shigella vaccines containing LPS can elicit antibodies capable of killing Shigella in a serotype-specific manner. Thus, to facilitate Shigella vaccine development, we have developed a serum bactericidal assay (SBA) specific for three Shigella serotypes that measures killing of target bacteria at multiple serum dilutions and in the presence of exogenous complement. The SBA has a high analytical throughput and uses simple technologies and readily available reagents. The SBA was characterized with human sera with bactericidal antibodies against S. flexneri 2a, S. flexneri 3a, and S. sonnei Purified LPS of a homologous serotype, but not a heterologous serotype, inhibited bacterial killing. Assessment of precision found median intra-assay precision to be 13.3% and median interassay precision to be 19 to 30% for the three serotypes. The SBA is linear, with slight deviations for samples with low (~40) killing indices. The SBA was sensitive enough to allow about 100-fold predilution of serum samples. Repeat assays yielded results with less than 2-fold deviations, indicating the robustness of the assay. Assay results from four different laboratories were highly comparable when normalized with a reference serum. The Shigella SBA, combined with a reference serum, should facilitate the development of Shigella vaccines across the field.IMPORTANCEShigella is an important cause of diarrhea worldwide, and efforts are ongoing to produce a safe and effective Shigella vaccine. Although a clear immune correlate of protection has not been established, antibodies with bactericidal capacity may provide one means of protecting against shigellosis. Thus, it is important to measure the functional capacity of antibodies, as opposed to only binding activity. This article describes a simple, robust, and high-throughput serum bactericidal assay capable of measuring Shigella-specific functional antibodies in vitro We show for the first time that this assay was successfully performed by multiple laboratories and generated highly comparable results, particularly when SBA titers were normalized using a reference standard. The serum bactericidal assay, along with a reference serum, should greatly facilitate Shigella vaccine development.

Development of recombinant vaccine candidate molecule against Shigella infection.

Shigellosis is an acute bacillary diarrheal disease caused by the gram negative bacillus Shigella. The existence of multiple Shigella serotypes and their growing resistance to antibiotics stress the urgent need for the development of vaccine that is protective across all serotypes. Shigella's IpaB antigen is involved in translocon pore formation, promotes bacterial invasion and induces apoptosis in macrophages. S. Typhi GroEL (Hsp 60) is the immunodominant antigen inducing both arms of immunity and has been explored as adjuvant in this study. The present study evaluates the immunogenicity and protective efficacy of recombinant IpaB domain-GroEL fusion protein in mice against lethal Shigella infection. The IpaB domain and GroEL genes were fused using overlap extension PCR and cloned in pRSETA expression vector. Fused gene was expressed in Escherichia coli BL-21 cells and the resulting 90 KDa fusion protein was purified by affinity chromatography. Intranasal (i.n.) immunization of mice with fusion protein increased the IgG and IgA antibody titers as compared to the group immunized with IpaB and GroEL and control PBS immunized group. Also IgG1 and IgG2a antibodies induced in fusion protein immunized mice were higher than co-immunized group. Significant increase in lymphocyte proliferation and cytokine levels (IFN-γ, IL-4 and IL-10), indicates induction of both Th1 and Th2 immune responses in both immunized groups. Immunization with fusion protein protected 90-95% of mice whereas 80-85% survivability was observed in co-immunized group against lethal challenge with S. flexneri, S. boydii and S. sonnei. Passive immunization conferred 60-70% protection in mice against all these Shigella species. Organ burden and histopathology studies also revealed significant decrease in lung infection as compared to the co-immunized group. Since IpaB is the conserved dominant molecule in all Shigella species, this study will lead to an ideal platform for the development of safe, efficacious and cost-effective recombinant vaccine against Shigella serotypes.

Recombinant heat shock protein 60 (Hsp60/GroEL) of Salmonella enterica serovar Typhi elicits cross-protection against multiple bacterial pathogens in mice.

Heat shock proteins (HSPs) or stress proteins are recognized as protective antigens against a wide range of bacterial diseases. Conservation of HSPs across different life forms also appears to contribute to the antigenicity of these proteins. Due to their high sequence homology, there exists an immunological cross-recognition between different bacterial species. In the present study, we evaluated the efficacy of recombinant GroEL of Salmonella enterica serovar Typhi as a vaccine candidate against various bacterial pathogens viz.; Shigella dysenteriae type I, Shigella flexneri, Shigella boydii, enteropathogenic Escherichia coli (EPEC), Klebsiella pneumoniae and Pseudomonas aeruginosa. In vitro serum bactericidal assay (SBA) with GroEL antisera showed 50-55% inhibition of cells of Shigella Spp., 65-75% of E. coli, 60-65% of K. pneumoniae, 45-50% of P. aeruginosa. In in vivo experiments, mice immunized with GroEL protein of S. Typhi showed 60-65% protection against S. flexneri, S. dysenteriae type I, S. boydii. Similarly 75-80% protection was observed against enteropathogenic E. coli, 70-80% against K. pneumoniae. 50% of mice survived the lethal infection against P. aeruginosa. Organ burden and histopathological studies also revealed significant reduction of bacterial infection. This study shows the cross-protective efficacy of recombinant GroEL of S. Typhi which could lead to the development of a single vaccine candidate protective against multiple bacterial pathogens.