Prokaryotic and eukaryotic dual-expression plasmid-mediated delivery of Campylobacter jejuni antigens by live-attenuated Salmonella: A strategy for concurrent Th1 and Th2 immune activation and protection in chickens.

Salmonella and Campylobacter are food-borne pathogens that significantly affect poultry production and are transmitted to humans. Long-term protection against these pathogens in chicken relies on a balanced Th1 and Th2 response. C. jejuni antigens were screened and a fusion antigen, including CadF + FlaA adhesin and flagellin antigenic fragments was developed and safely delivered by low-endotoxicity S. Typhimurium through pJHL270, a dual-expression plasmid featuring prokaryotic (Ptrc) and eukaryotic (CMV) promoters. Antigen expression in Salmonella and host cells was confirmed by western blotting and IFA. The vaccine construct JOL2999, triggered significant increases in IgY, IgA antibodies, CD4+ and CD8+ T cells, indicating humoral, mucosal, and cell-mediated responses against both pathogens. Elevations in pro-inflammatory cytokines TNFα, INF-γ, IL-2, and IL-4 and MHC I and II cell populations further suggest simultaneous Th1 and Th2 immune activation. Reduced pathogen load and histopathological inflammatory signs in vital organs upon challenge confirmed the protective efficacy in chickens.

Assessment of environmental safety and protective efficacy of O-antigen deficient DIVA capable Salmonella Enteritidis against chicken salmonellosis.

In this study, we incorporated deletion of the O-antigen ligase gene to an attenuated Salmonella Enteritidis (SE) strain, JOL919 (SE PS; Δlon ΔcpxR), using the Lambda-Red recombination method and evaluated the safety and immunological aspects of the novel genotype, JOL2381 (SE VS: Δlon, ΔcpxR, ΔrfaL). Assessment of fecal shedding and organ persistence following administration via oral and IM routes revealed that the SE VS was safer than its parent strain, SE PS. Immunological assays confirmed that immunization via the oral route with SE PS was superior to the SE VS. However, chickens immunized with SE PS and SE VS strains via the IM route showed higher humoral and cell-mediated immune responses. Compared to PBS control, the IM route of immunization with SE VS resulted in a higher IgY antibody titer and expansion of CD4+ and CD8+ T-cell populations, which resulted in the clearance of Salmonella from the liver and splenic tissues. Furthermore, deletion of the O-antigen ligase gene caused lower production of LPS-specific antibodies in the host, promoting DIVA functionality and making it a plausible candidate for field utilization. Due to significant protection, high attenuation, and environmental safety concerns, the present SE VS strain is an ideal choice to prevent chicken salmonellosis and ensure public health.

Tumor-targeted delivery of lnc antisense RNA against RCAS1 by live-attenuated tryptophan-auxotrophic Salmonella inhibited 4T1 breast tumors and metastasis in mice.

Emerging chemo- and radiotherapy resistance exacerbated the cancer risk and necessitated novel treatment strategies. Although RNA therapeutics against pro-oncogenic genes are highly effective, tumor-specific delivery remains a barrier to the implementation of this valuable tool. In this study, we report a tryptophan-auxotrophic Salmonella typhimurium strain as an onco-therapeutic delivery system with tumor-targeting ability using 4T1 mice breast-cancer model. The receptor-binding cancer antigen expressed on SiSo cell (RCAS1) is a cancer-specific protein that induces the apoptosis of peripheral lymphocytes and confers tumor immune evasion. We designed a long non-coding antisense-RNA against RCAS1 (asRCAS1) and delivered by Salmonella using a non-antibiotic, auxotrophic-selective, eukaryotic expression plasmid, pJHL204. After in vivo tumor-to-tumor passaging, the JOL2888 (ΔtrpA, ΔtrpE, Δasd + asRCAS1) strain exhibited high sustainability in tumors, but did not last in healthy organs, thereby demonstrating tumor specificity and safety. RCAS1 inhibition in the tumor was confirmed by western blotting and qPCR. In mice, JOL2888 treatment reduced tumor-associated macrophages, improved the T cell population, elicited cell-mediated immunity, and suppressed cancer-promoting genes. Consequently, the JOL2888 treatment significantly decreased the tumor volume by 80%, decreased splenomegaly by 30%, and completely arrested lung metastasis. These findings highlight the intrinsic tumor-targeting ability of tryptophan-auxotrophic Salmonella for delivering onco-therapeutic macromolecules.

Inflammation-Related Immune-Modulatory SLURP1 Prevents the Proliferation of Human Colon Cancer Cells, and Its Delivery by Salmonella Demonstrates Cross-Species Efficacy against Murine Colon Cancer.

This study investigates the anticancer properties of the α7-nAChR antagonist SLURP1 with a specific focus on its effect as an inflammation modulator on human colorectal cancer cell lines Caco2, Colo320DM, and H508 cells. The investigation includes the evaluation of cell cycle arrest, cell migration arrest, endogenous expression of SLURP1 and related proteins, calcium influx, and inflammatory responses. The results demonstrate that SLURP1 not only inhibits cell proliferation but also has the potential to arrest the cell cycle at the G1/S interface. The impact of SLURP1 on cell cycle regulation varied among cell lines, with H508 cells displaying the strongest response to exogenous SLURP1. Additionally, SLURP1 affects the nuclear factor kappa B expression and effectively reverses inflammatory responses elicited by purified lipopolysaccharides in H508 and Caco2 cells. This study further confirmed the expression of human SLURP1 by Salmonella, under Ptrc promoter, through Western blot analysis. Moreover, Salmonella secreting SLURP1 revealed a significant tumor regression in a mouse CT26 tumor model, suggesting the cross-species anticancer potential of human SLURP1. However, further investigations are required to fully understand the mechanisms underlying SLURP1's ability to prevent cancer proliferation and its protective function in humans.

Salmonella delivers H9N2 influenza virus antigens via a prokaryotic and eukaryotic dual-expression vector and elicits bivalent protection against avian influenza and fowl typhoid.

The H9N2 avian influenza virus significantly affects the health of poultry and humans. We identified a prokaryotic and eukaryotic dual-expression vector system, pJHL270, that can provide simultaneous MHC class I and II stimulation of the host immune system, and we designed vaccine antigens by selecting the consensus HA1 sequence and M2e antigens from H9N2 virus circulating in South Korea from 2000 to 2021. The genes were cloned into the pJHL270 vector, and the cloned plasmid was delivered by a live-attenuated Salmonella Gallinarum (SG) strain. The immunity and protective efficacy of the SG-based H9N2 vaccine construct, JOL2922, against avian influenza and fowl typhoid (FT) were evaluated. The Ptrc and CMV promoters conferred antigen expression in prokaryotic and eukaryotic cells to induce balanced Th-1/Th-2 immunity. Chickens immunized with JOL2922 yielded high antigen-specific humoral and mucosal immune responses. qRT-PCR revealed that the strain generated polyfunctional IFN-γ and IL-4 secretion in immunized chickens. Furthermore, a FACS analysis showed increased CD3CD4+ and CD3CD8+ T-cell subpopulations following immunization. Peripheral Blood Mononuclear Cells (PBMCs) harvested from the immunized chickens significantly increased MHC class I and II expression, 3.5-fold and 2.5-fold increases, respectively. Serum collected from the immunized groups had an evident hemagglutinin inhibition titer of ≥6 log2. Immunization reduced the lung viral titer by 3.8-fold within 5 days post-infection. The strain also generated SG-specific humoral and cellular immune responses. The immunized birds all survived a virulent SG wild-type challenge. In addition, the bacterial burden was reduced by 2.7-fold and 2.1-fold in spleen and liver tissue, respectively, collected from immunized chickens. Our data indicate that an attenuated SG strain successfully delivered the dual-expression vector system and co-stimulated MHC class I and II antigen presentation pathways via exogenous and endogenous antigen presentation, thereby triggering a balanced Th-1/Th-2-based immune response and conferring effective protection against avian influenza and FT.

An mRNA-Based Multiple Antigenic Gene Expression System Delivered by Engineered Salmonella for Severe Fever with Thrombocytopenia Syndrome and Assessment of Its Immunogenicity and Protection Using a Human DC-SIGN-Transduced Mouse Model.

Currently, there are no commercial vaccines or therapeutics against severe fever with thrombocytopenia syndrome (SFTS) virus. This study explored an engineered Salmonella as a vaccine carrier to deliver a eukaryotic self-mRNA replicating vector, pJHL204. This vector expresses multiple SFTS virus antigenic genes for the nucleocapsid protein (NP), glycoprotein precursor (Gn/Gc), and nonstructural protein (NS) to induce host immune responses. The engineered constructs were designed and validated through 3D structure modeling. Western blot and qRT-PCR analyses of transformed HEK293T cells confirmed the delivery and expression of the vaccine antigens. Significantly, mice immunized with these constructs demonstrated a cell-mediated and humoral response as balanced Th1/Th2 immunity. The JOL2424 and JOL2425 delivering NP and Gn/Gc generated strong immunoglobulin IgG and IgM antibodies and high neutralizing titers. To further examine the immunogenicity and protection, we utilized a human DC-SIGN receptor transduced mouse model for SFTS virus infection by an adeno-associated viral vector system. Among the SFTSV antigen constructs, the construct with full-length NP and Gn/Gc and the construct with NP and selected Gn/Gc epitopes induced robust cellular and humoral immune responses. These were followed by adequate protection based on viral titer reduction and reduced histopathological lesions in the spleen and liver. In conclusion, these data indicate that recombinant attenuated Salmonella JOL2424 and JOL2425 delivering NP and Gn/Gc antigens of SFTSV are promising vaccine candidates that induce strong humoral and cellular immune responses and protection against SFTSV. Moreover, the data proved that the hDC-SIGN transduced mice as a worthy tool for immunogenicity study for SFTSV.

The impact of lipid A modification on biofilm and related pathophysiological phenotypes, endotoxicity, immunogenicity, and protection of Salmonella Typhimurium.

This study presents the engineering of a less endotoxic Salmonella Typhimurium strain by manipulating the lipid-A structure of the lipopolysaccharide (LPS) component. Salmonella lipid A was dephosphorylated by using lpxE from Francisella tularensis. The 1-phosphate group from lipid-A was removed selectively, resulting in a close analog of monophosphoryl lipid A. We observed a significant impact of ∆pagL on major virulence factors such as biofilm formation, motility, persistency, and immune evasion. In correlation with biofilm and motility retardation, adhesion and invasion were elevated but with reduced intracellular survival, a favorable phenotype prospect of a vaccine strain. Western blotting and silver staining confirmed the absence of the O-antigen and truncated lipid-A core in the detoxified Salmonella mutant. In vitro and in vivo studies demonstrated that the dephosphorylated Salmonella mutant mediated lower pro-inflammatory cytokine secretion than the wild-type strain. The vaccine strains were present in the spleen and liver for five days and were cleared from the organs by day seven. However, the wild-type strain persisted in the spleen, liver, and brain, leading to sepsis-induced death. Histological evaluations of tissue samples further confirmed the reduced endotoxic activity of the detoxified Salmonella mutant. The detoxification strategy did not compromise the level of protective immunity, as the vaccine strain could enhance humoral and cellular immune responses and protect against the wild-type challenge in immunized mice.

Novel pro-and eukaryotic expression plasmid expressing omicron antigens delivered via Salmonella elicited MHC class I and II based protective immunity.

The latest omicron variants are emerging with mutations in the receptor binding domain (RBD) that confer immune evasion and resistance against current vaccines. Such variants have raised the peril of future vaccine effectiveness, as leading vaccines target the spike protein. Type-IV hypersensitivity, and other ailments due to the dominant Th1 response by leading vaccines, is also to be resolved. Therefore, vaccine that target diverse SARS-CoV-2 proteins and provide broad-spectrum protection and a balanced Th1 and Th2 response is an indispensable armament against the pandemic. In that prospect, a novel dual expression plasmid pJHL270 was developed and demonstrated the expression of omicron antigens exogenously from Salmonella and endogenously in the host cells. The simultaneous activation of MHC class I and II molecules culminated in a balanced Th1 and Th2 response, which was evident through the upsurge of IgG, IgA antibodies, IgG2a/IgG1 ratio, cytokine responses and CD4+, CD8+ T-lymphocytes. The level of CD44+ cells showed the trigger for Th1 and Th2 balance and memory-cell activation for long-lasting immunity. The level of IFN-γ + cells and neutralizing antibodies signifies the anti-viral response. The vaccine protected the hamsters from BA.5 and BA.2.75 omicron viral-challenge, exhibited a significant reduction in lung viral-load and histopathological lesions. In addition to two-way antigen expression and bilateral immune elicitation, this Salmonella-based vaccine delivery system can be prospectively applied to humans and a broad range of animals as a convenient alternative to viral and chemical vaccine delivery approaches.

Screening of lipid-A related genes and development of low-endotoxicity live-attenuated Salmonella gallinarum by arnT deletion that elicits immune responses and protection against fowl typhoid in chickens.

In the present study, lipid-A gene mutants of Salmonella gallinarum (SG) were screened, and the arnT mutant exhibited optimal acidic and oxidative-stress and macrophage-survival. Modifying lipid-A by arnT-deletion resulted in significantly reduced endotoxicity, virulence, and mortality. Therefore, the arnT-deleted vaccine-candidate strain JOL2841 was constructed and demonstrated to be safe due to appropriate clearance by the chicken immune system. The reduced-endotoxicity of JOL2841 was evident from the downregulation of TNFα and IL-1ß inflammatory cytokines, no inflammatory signs in organ gross-examination, and histopathological analysis. The IgY and IgA antibody titres, CD4, and CD8 T-cell population improvements, and IL-4, IL-2, and INFγ expression decipher the profound Th2 and Th1 immunogenicity. Consequently, JOL2841 exhibited prominent protection against wild-type SG challenge, as revealed by organ pathogen-load determination, organ gross-examination, and histopathological examination. Overall, the study represented the first report of arnT deficient SG resulted in negligible endotoxicity, low-virulence, safety and coordinated elicitation of humoral and cell-mediated immune response in chickens.

Development and evaluation of a mouse model susceptible to severe fever with thrombocytopenia syndrome virus by rAAV-based exogenous human DC-SIGN expression.

Experimental animal model is indispensable to evaluate the prophylactic and therapeutic candidates against severe fever with thrombocytopenia syndrome virus (SFTSV). To develop a suitable mouse model for SFTSV infection, we delivered human dendritic cell-specific ICAM-3-grabbing non-integrin (hDC-SIGN) by adeno-associated virus (AAV2) and validated its susceptibility for SFTSV infection. Western blot and RT-PCR assays confirmed the expression of hDC-SIGN in transduced cell lines and a significantly increased viral infectivity was observed in cells expressing hDC-SIGN. The C57BL/6 mice transduced with AAV2 exhibited a stable hDC-SIGN expression in the organs for 7 days. Upon SFTSV challenge with 1 × 105 FAID50, the mice transduced with rAAV-hDC-SIGN showed a 12.5% mortality and reduced platelet and white blood cell count in accordance with higher viral titer than control group. Liver and spleen samples collected from the transduced mice had pathological signs similar to the IFNAR-/- mice with severe SFTSV infection. Collectively, the rAAV-hDC-SIGN transduced mouse model can be used as an accessible and promising tool for studying the SFTSV pathogenesis and pre-clinical evaluation of vaccines and therapeutics against the SFTSV infection.

Salmonella-mediated oral delivery of multiple-target vaccine constructs with conserved and variable regions of SARS-CoV-2 protect against the Delta and Omicron variants in hamster.

Since the emergence of the pandemic COVID19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of vaccines has been the prime strategy to control the disease transmission. Most of the developed vaccines target the spike protein, however, the emerging variants have alterations, particularly at the same region which may pose resistance to neutralizing antibodies. In this study, we explored the variable and conserved regions of SARS-CoV-2 as a potential inclusion in a multiple-target vaccine with the exploitation of Salmonella-based vector for oral mRNA vaccine against Delta and Omicron variants. Increased IgG and IgA levels imply the induction of humoral response and the CD4+, CD8+ and IFN-γ+ sub-population level exhibits cell-mediated immune responses. The degree of CD44+ cells indicates the induction of memory cells corresponding to long-term immune responses. Furthermore, we assessed the protective efficacy of the vaccines against the Delta and Omicron variants in the hamster model. The vaccine constructs induced neutralizing antibodies and protected the viral-challenged hamsters with significant decrease in lung viral load and reduced histopathological lesions. These results reinforce the use of the conserved and variable regions as potential antigen targets of SARS-CoV-2 as well as the exploitation of bacteria-mediated delivery for oral mRNA vaccine development.

Efficacy of a Novel Multiepitope Vaccine Candidate against Foot-and-Mouth Disease Virus Serotype O and A.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease in cloven-hoofed animals. To prevent the spread of FMD virus (FMDV), traditional inactivated vaccines are used to immunize susceptible animals in disease-endemic countries. However, the inactivated FMD vaccine has several limitations, including safety concerns. To overcome these limitations, subunit proteins have been studied as alternative vaccine candidates. In this study, we designed two multiepitope recombinant proteins (OVM and AVM) containing antigenic sites (residue of VP1 132-162 and residue of VP1 192-212) of three topotypes of FMDV serotype O or three topotypes of FMDV serotype A. Each recombinant protein was efficiently expressed in Escherichia coli with high solubility, and the immunogenicity and protective efficacy of the proteins as FMD vaccine candidates were evaluated. The results showed that OVM and AVM emulsified with ISA201 adjuvant induced effective antigen-specific humoral and cell-mediated immune responses and successfully protected mice from O/Jincheon/SKR/2014, O/VET/2013, and A/Malaysia/97 viruses. In addition, intramuscular immunization of pigs with the OVM and AVM emulsified with ISA201 elicited effective levels of neutralizing antibodies to the viruses with homologous epitopes. Importantly, OVM-AVM emulsified with CAvant®SOE-X adjuvant conferred 100% protection against the O/Jincheon/SKR/2014 virus with homologous residues and 75% protection against A/SKR/GP/2018 with heterologous residues. The results presented in this study suggest that the combination of OVM and AVM protein with an effective adjuvant could yield an effective and safe vaccine candidate for the prevention and control of foot-and-mouth disease. In addition, our results provide a vaccine platform that can safely, cost-efficiently, and rapidly generate protective vaccine candidates against diverse FMDVs.

Safety assessment of compliant, highly invasive, lipid A-altered, O-antigen-defected Salmonella strains as prospective vaccine delivery systems.

In the present study, two prospective Salmonella delivery strains, JOL2782 and JOL2837, were developed by gene deletions of lon and cpxR, which are related to cellular adhesion and intracellular survival. Additionally, sifA deletion was introduced for JOL2782, which confers immune susceptibility and improves antigen delivery. Similarly, the rfaL deletion and lpxE substitution for pagL were accomplished in JOL2837 to reduce virulence and endotoxicity. Thus, enhanced adhesion and invasion and reduced intracellular survival were attained. Furthermore, aspartic acid auxotrophic (asd) was deleted to impose Darwinian selection on retention of the foreign antigen-expressing plasmid. Both delivery strains induced sufficient cytokine expression, but the level was significantly lower than that of the wild-type strain; the lowest cytokine expression was induced by the JOL2837 strain, indicating reduced endotoxicity. In parallel, IgG production was significantly enhanced by both delivery strains. Thus, the innate and adaptive immunogenicity of the strains was ensured. The environmental safety of these strains was ascertained through faecal dissemination assays. The nonpathogenicity of these strains to the host was confirmed by body weight monitoring, survival assays, and morphological and histological assessments of the vital organs. The in vitro assay in murine and human cell lines and in vivo safety assessments in mice suggest that these novel strains possess safety, invasiveness, and immunogenicity, making them ideal delivery strains. Overall, the results clearly showed that strain JOL2782 with sifA deletion had higher invasiveness, demonstrating superior vaccine deliverability, while JOL2837 with lpxE substitution for pagL and rfaL deletion had outstanding safety potential with drastically abridged endotoxicity.

Prospective lipid-A altered live attenuated Salmonella Gallinarum confers protectivity, DIVA capability, safety and low endotoxicity against fowl typhoid.

The present study describes creating an attenuated Salmonella Gallinarum (SG) strain with reduced endotoxicity to prevent fowl typhoid. The strain was attenuated by deleting the lon, cpxR, and rfaL virulence-related genes. Endotoxicity was reduced by deleting the pagL open reading frame and replacing it with the lpxE gene derived from Francisella tularencis. Both events, (1) deletion of the pagL and (2) introduction of the lpxE genes, conferred reduced endotoxicity by detoxifying the lipid A structure. The detoxified SG strain (SGVSdt) was well tolerated in 7-day-old chicks when administered orally at 1 × 108 CFU/bird and in 14-day-old birds administered 1 × 107 CFU/bird subcutaneously. Parenteral immunization of detoxified vaccine strain was completely safe in birds and free of environmental contamination. Subcutaneous immunization conferred disease protection and induced humoral and cell-mediated immune responses marked by Th1-skewed patterns similar to those produced by the commercial SG9R vaccine strain. Compared with the SG9R-based vaccine, the SGVSdt construct generated significantly fewer inflammatory TNF-α responses while significantly inducing IFN-γ cytokine levels as an indication of an adaptive antibacterial response. The differentiating infected from vaccinated animals (DIVA) capability was on par with the predecessor SGVS. This study presents an appealing biological strategy to minimize lipid A-mediated endotoxicity without compromising protective efficacy against the SG challenge. Reduced endotoxicity permits the utilization of higher inoculation doses to maximize protection against fowl typhoid.

Assessing an O-antigen deficient, live attenuated Salmonella Gallinarium strain that is DIVA compatible, environmentally safe, and protects chickens against fowl typhoid.

The objective of the present study was to create a highly attenuated, safe Salmonella Gallinarium (SG) vaccine strain for chicken vaccination against fowl typhoid (FT) diseases. The SG vaccine strain (SGVS) consists of three virulence-related gene deletions, namely, lon, cpxR, and rfaL. The parent strain (SGPS) with Δlon ΔcpxR genotype was utilized as the host strain for in-frame rfaL gene deletion by lambda red recombination. The SGVS was highly attenuated with improved environmental safety by zero fecal contamination beyond seven days for both oral and intramuscular immunization routes. Upon inoculation into 1-month-old young chicken, no vaccine-induced adverse behaviors were observed and did not cause a chronic state of infection as the SG wild-type strain did. Immunization of chicken elicited both humoral and cell-mediated immune responses demarcated by, IgY antibody assessment, T-cell responses in peripheral blood mononuclear cells, and the induction of immunomodulatory cytokines, IFN-γ, IL-2, IL-12, and IL-4 to resemble both Th1 and Th2 type of immune responses. The immunological assessment revealed a high level of efficacy of the SGVS when inoculated via the IM route than the oral route. The strain was less cytotoxic with reduced cytotoxicity on chicken macrophages and was DIVA capable with minimum reactivity of immunized serum with purified SG lipopolysaccharides. The challenge study could generate 70% protection in chicken for SGVS, whereas no birds were protected in the PBS challenged group. The protection levels were evident in histopathological assessment of spleen and liver specimens and also the external appearance of the spleen with reduced lesions on immunized groups. Further experiments may be warranted to dose and route optimization for further increase in the protection level derived by present SGVS.

A Simplified SARS-CoV-2 Mouse Model Demonstrates Protection by an Oral Replicon-Based mRNA Vaccine.

A mouse model of SARS-CoV-2 that can be developed in any molecular biology lab with standard facilities will be valuable in evaluating drugs and vaccines. Here we present a simplified SARS-CoV-2 mouse model exploiting the rapid adenoviral purification method. Mice that are sensitive to SARS-CoV-2 infection were generated by transducing human angiotensin-converting enzyme 2 (hACE2) by an adenovirus. The expression kinetics of the hACE2 in transduced mice were assessed by immunohistochemistry, RT-PCR, and qPCR. Further, the ability of the hACE2 to support viral replication was determined in vitro and in vivo. The hACE2 expression in the lungs of mice was observed for at least nine days after transduction. The murine macrophages expressing hACE2 supported viral replication with detection of high viral titers. Next, in vivo studies were carried out to determine viral replication and lung disease following SARS-CoV-2 challenge. The model supported viral replication, and the challenged mouse developed lung disease characteristic of moderate interstitial pneumonia. Further, we illustrated the utility of the system by demonstrating protection using an oral mRNA vaccine. The multicistronic vaccine design enabled by the viral self-cleaving peptides targets receptor binding domain (RBD), heptad repeat domain (HR), membrane glycoprotein (M) and epitopes of nsp13 of parental SARS-CoV-2. Further, Salmonella and Semliki Forest virus replicon were exploited, respectively, for gene delivery and mRNA expression. We recorded potent cross-protective neutralizing antibodies in immunized mice against the SARS-CoV-2 delta variant. The vaccine protected the mice against viral replication and SARS-CoV-2-induced weight loss and lung pathology. The findings support the suitability of the model for preclinical evaluation of anti-SARS-CoV-2 therapies and vaccines. In addition, the findings provide novel insights into mRNA vaccine design against infectious diseases not limiting to SARS-CoV-2.

Highly feasible immunoprotective multicistronic SARS-CoV-2 vaccine candidate blending novel eukaryotic expression and Salmonella bactofection.

Introduction: The emergence of SARS-CoV-2 variants has raised concerns on future vaccine efficacy as most vaccines target only the spike protein. Hence, vaccines targeting multiple SARS-CoV-2 proteins will offer broader protection and improve our preparedness to combat the pandemic. Objectives: The study aimed to develop a novel vaccine strategy by combining a eukaryotic vector expressing multiple SARS-CoV-2 genes and Salmonella-mediated in vivo DNA delivery. Methods: The eukaryotic vector was designed to function as a DNA-launched RNA replicon in a self-replicating and self-amplifying mRNA mechanism. By exploiting the self-cleaving peptide, P2A, we fused four SARS-CoV-2 targets, including receptor-binding domain (RBD), heptad repeat domain (HR), membrane protein (M) and epitopes of nsp13, in a single open reading frame. Western blot and immunofluorescence assays were used to determine protein expression. In mice, the vaccine's safety and immunogenicity were investigated. Results: Western blot analysis revealed co-expression all four proteins from the vaccine construct, confirming the efficiency of Salmonella-mediated gene delivery and protein expression. The vaccine candidate was safe and elicited robust antigen-specific antibody titers in mice, and a recall response from splenocytes revealed induction of strong cell-mediated immunity. Flow cytometry demonstrated an increase in sub-populations of CD4+ and CD8+ T cells with the highest CD4+ and CD8+ T cells recorded for HR and RBD, respectively. Overall, humoral and cellular immune response data suggested the induction of both Th1 and Th2 immunity with polarization towards an antiviral Th1 response. We recorded a potent SARS-CoV-2 neutralizing antibody titers in the immunized mice sera. Conclusions: The Salmonella bactofection ensured optimum in vivo gene delivery, and through a P2A-enabled efficient multicistronic expression, the vaccine candidate elicited potent anti-SARS-CoV-2 immune responses. These findings provide important insight into development of an effective multivalent vaccine to combat SARS-CoV-2 and its variants.

Bacteria-enabled oral delivery of a replicon-based mRNA vaccine candidate protects against ancestral and delta variant SARS-CoV-2.

The ongoing severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) evolution has resulted in many variants, contributing to the striking drop in vaccine efficacy and necessitating the development of next-generation vaccines to tackle antigenic diversity. Herein we developed a multivalent Semliki Forest virus replicon-based mRNA vaccine targeting the receptor binding domain (RBD), heptad repeat domain (HR), membrane protein (M), and epitopes of non-structural protein 13 (nsp13) of SARS-CoV-2. The bacteria-mediated gene delivery offers the rapid production of large quantities of vaccine at a highly economical scale and notably allows needle-free mass vaccination. Favorable T-helper (Th) 1-dominated potent antibody and cellular immune responses were detected in the immunized mice. Further, immunization induced strong cross-protective neutralizing antibodies (NAbs) against the B.1.617.2 delta variant (clade G). We recorded a difference in induction of immunoglobulin (Ig) A response by the immunization route, with the oral route eliciting a strong mucosal secretory IgA (sIgA) response, which possibly has contributed to the enhanced protection conferred by oral immunization. Hamsters immunized orally were completely protected against viral replication in the lungs and the nasal cavity. Importantly, the vaccine protected the hamsters against SARS-CoV-2-induced pneumonia. The study provides proof-of-principle findings for the development of a feasible and efficacious oral mRNA vaccine against SARS-CoV-2 and its variants.

Immunization of chickens with Salmonella gallinarium ghosts expressing Salmonella Enteritidis NFliC-FimAC and CD40LC fusion antigen enhances cell-mediated immune responses and protects against wild-type challenges with both species.

This study describes the construction and immunological characterization of a novel Salmonella gallinarium ghost vaccine to protect against S. gallinarium (SG) and S. Enteritidis (SE) serotypes. The SG ghost was designed to express N-terminus FliC (D0-D1 domain) and FimA retrieved from the SE genome, and the receptor-binding domain (RBD) of CD40L from the chicken as a single fusion construct. The construct was built in pJHL184, a phage lysis gene E-mediated ghost plasmid and the expression was confirmed by western blot resulting in an 85-kDa band. Chicken immunization was conducted by intramuscular route with SG ghost FliC-FimA-CD40L, vector control, or PBS alone in a prime-boost schedule. Antibody responses, cell-mediated immune responses (CMI), and cytokine induction was assessed in chicken demonstrating significantly high levels of IgY, CMI, cytokine responses in ghost immunized group delivering partial protection against SG wild type challenge and near complete protection against SE challenge wild type challenge.

Eukaryotic expression system complemented with expressivity of Semliki Forest Virus's RdRp and invasiveness of engineered Salmonella demonstrate promising potential for bacteria mediated gene therapy.

This study describes an efficient eukaryotic expression system (pJHL204) built into the Salmonella delivery system to enhance the essential efficacy and effectiveness of conventional DNA therapy. The expression system utilizes RNA-dependent RNA polymerase activity (RdRp) of Semiliki Forest Virus attributing to dramatic antigen expression by cytoplasmic mRNA amplification. Functional characterization of the pJHL204 by in vitro and in vivo transfection studies revealed the improved expression of mRNA at least 150 folds than the RdRp mutant plasmid under in vitro conditions. Using green fluorescence protein (GFP) and mCherry as bait proteins this system was extensively characterized for plasmid delivery capacity, antigen expression, and safety using in vivo and in vitro models by employing flow cytometry, fluorescence microscopy, and immunohistochemical staining. Employment of Salmonella as a carrier significantly extends plasmid in vivo survivability and prolongs the effective duration until the elimination of the Salmonella carrier strain in the host. The strategy can be easily adapted for P2A connected multiple antigen delivery in a single vector system due to the significantly high cargo capacity of Salmonella. A mouse challenge study was carried out utilizing P2A connected H1N1 hemagglutinin (HA) and neuraminidase (NA) via the Salmonella carrier strain JOL2500 significantly reduced viral activity and protected mice against the H1N1 challenge and demonstrates potential to redefine in vivo DNA therapy as a reliable and safe system to treat human diseases using useful microbes like Salmonella.