Comparative transcriptome analysis of acne vulgaris, rosacea, and hidradenitis suppurativa supports high-dose dietary zinc as a therapeutic agent.

Acne vulgaris, rosacea, and hidradenitis suppurativa are enduring inflammatory skin conditions that frequently manifest with akin clinical attributes, posing a considerable challenge for their distinctive diagnosis. While these conditions do exhibit certain resemblances, they also demonstrate distinct underlying pathophysiological mechanisms and treatment modalities. Delving into both the molecular parallels and disparities among these three disorders can yield invaluable insights for refined diagnostics, effective management, and targeted therapeutic interventions. In this report, we present a comparative analysis of transcriptomic data across these three diseases, elucidating differentially expressed genes and enriched pathways specific to each ailment, as well as those shared among them. Specifically, we identified multiple zinc-binding proteins (SERPINA1, S100A7, S100A8, S100A9 and KRT16) as consistently highly upregulated genes across all three diseases. Our hypothesis suggests that these proteins could bind and sequester zinc, potentially leading to localized zinc deficiency and heightened inflammation. We identified high-dose dietary zinc as a promising therapeutic approach and confirmed its effectiveness through validation in an acne mouse model.

Intranasally administered protein coated chitosan nanoparticles encapsulating influenza H9N2 HA2 and M2e mRNA molecules elicit protective immunity against avian influenza viruses in chickens.

Chitosan nanoparticles (CNPs) represent an efficient vaccination tool to deliver immunogenic antigens to the antigen-presenting cells (APCs), which subsequently stimulate protective immune responses against infectious diseases. Herein, we prepared CNPs encapsulating mRNA molecules followed by surface coating with conserved H9N2 HA2 and M2e influenza proteins. We demonstrated that CNPs efficiently delivered mRNA molecules into APCs and had effectively penetrated the mucosal barrier to reach to the immune initiation sites. To investigate the potential of CNPs delivering influenza antigens to stimulate protective immunity, we intranasally vaccinated chickens with empty CNPs, CNPs delivering HA2 and M2e in both mRNA and protein formats (CNPs + RNA + Pr) or CNPs delivering antigens in protein format only (CNPs + Pr). Our results demonstrated that chickens vaccinated with CNPs + RNA + Pr elicited significantly (p < 0.05) higher systemic IgG, mucosal IgA antibody responses and cellular immune responses compared to the CNPs + Pr vaccinated group. Consequently, upon challenge with either H7N9 or H9N2 avian influenza viruses (AIVs), efficient protection, in the context of viral load and lung pathology, was observed in chickens vaccinated with CNPs + RNA + Pr than CNPs + Pr vaccinated group. In conclusion, we show that HA2 and M2e antigens elicited a broad spectrum of protection against AIVs and incorporation of mRNAs in vaccine formulation is an effective strategy to induce superior immune responses.

Oral immunization with an attenuated Salmonella Gallinarum encoding the H9N2 haemagglutinin and M2 ectodomain induces protective immune responses against H9N2 infection in chickens.

H9N2, a low pathogenic avian influenza virus, causes significant economic losses in the poultry industry worldwide. Herein, we describe the construction of an attenuated Salmonella Gallinarum (SG) strain for expression and delivery of H9N2 haemagglutinin (HA) 1 (SG-HA1), HA2 (SG-HA2) and/or the conserved matrix protein 2 ectodomain (SG-M2e). We demonstrated that recombinant SG strains expressing HA1, HA2 and M2e antigens were immunogenic and safe in a chicken model. Chickens (n = 8) were vaccinated once orally with SG alone, SG-HA1, SG-HA2, SG-M2e, or mixture of SG-HA1, SG-HA2 and SG-M2e, or vaccinated once intramuscularly with an oil-adjuvant inactivated H9N2 vaccine. Our results demonstrated that vaccination with SG mutants encoding influenza antigens, administered individually or as a mixture, elicited significantly (P < 0.05) greater antigen-specific humoral and cell-mediated immune responses in chickens compared with those vaccinated with SG alone. A conventional H9N2 vaccine induced significantly (P < 0.05) greater HA1 and HA2 antibody responses than SG-based H9N2 vaccine strains, but significantly (P < 0.05) less robust M2e-specific responses. Upon challenge with the virulent H9N2 virus on day 28 post-vaccination, chickens vaccinated with either the SG-based H9N2 or conventional H9N2 vaccines exhibited comparable lung inflammation and viral loads, although both were significantly lower (P < 0.05) than in the group vaccinated with SG alone. In conclusion, our results showed that SG-based vaccination stimulated efficient immune responses against virulent H9N2. Further studies are needed to fully develop this approach as a preventive strategy for low pathogenic avian influenza viruses affecting poultry. RESEARCH HIGHLIGHTS S. gallinarum expressing HA1, HA2 and M2e antigens are immunogenic and safe. Salmonella has dual function of acting as a delivery system and as a natural adjuvant. Vaccine constructs elicit specific humoral and cell-mediated immune responses.

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

Fowl typhoid (FT), a septicemic disease caused by Salmonella 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 (p < 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 (105 TCID50) at 28th day post-vaccination, chickens that received a co-mix of JOL2074 plus JOL2076 strains exhibited significantly (p < 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.

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

Attenuated Salmonella 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 Salmonella 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 Salmonella 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 Salmonella (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.

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.

Fowl typhoid (FT), a septicemic disease caused by Salmonella Gallinarum (SG), and infectious bronchitis (IB) are two economically important avian diseases that affect poultry industry worldwide. Herein, we exploited a live attenuated SG mutant, JOL967, to deliver spike (S) protein 1 of IB virus (V) to elicit protective immunity against both FT and IB in chickens. The codon optimized S1 nucleotide sequence was cloned in-frame into a prokaryotic constitutive expression vector, pJHL65. Subsequently, empty pJHL65 or recombinant pJHL65-S1 plasmid was electroporated into JOL967 and the resultant clones were designated as JOL2068 and JOL2077, respectively. Our results demonstrated that the chickens vaccinated once orally with JOL2077 elicited significantly (p < 0.05) higher IBV-specific humoral and cell-mediated immunity compared to JOL2068 and PBS control groups. Consequently, on challenge with the virulent IBV strain at 28th day post-vaccination, JOL2077 vaccinated birds displayed significantly (p < 0.05) lower inflammatory lesions in virus-targeted tissues compared to control groups. Furthermore, 33.3% (2 of 6) of birds vaccinated with JOL2077 vaccine had shown virus recovery from tracheal tissues compared to 100% (6 of 6) recovery obtained in both the control groups. Against wild-type SG lethal challenge, both JOL2077 and JOL2068 vaccinated groups exhibited only 10% mortality compared to 80% mortality observed in PBS control group. In conclusion, we show that JOL2077 can induce efficient IBV- and carrier-specific protective immunity and can act as a bivalent vaccine against FT and IB. Further studies are warranted to investigate the potential of JOL2077 vaccine in broiler and young layer birds.

Bacterial ghosts as adjuvants: mechanisms and potential.

Bacterial ghosts (BG) are empty cell envelopes derived from Gram-negative bacteria. They contain many innate immunostimulatory agonists, and are potent activators of a broad range of cell types involved in innate and adaptive immunity. Several considerable studies have demonstrated the effectiveness of BG as adjuvants as well as their ability to induce proinflammatory cytokine production by a range of immune and non-immune cell types. These proinflammatory cytokines trigger a generalized recruitment of T and B lymphocytes to lymph nodes that maximize the chances of encounter with their cognate antigen, and subsequent elicitation of potent immune responses. The plasticity of BG has allowed for the generation of envelope-bound foreign antigens in immunologically active forms that have proven to be effective vaccines in animal models. Besides their adjuvant property, BG also effectively deliver DNA-encoded antigens to dendritic cells, thereby leading to high transfection efficiencies, which subsequently result in higher gene expressions and improved immunogenicity of DNA-based vaccines. In this review, we summarize our understanding of BG interactions with the host immune system, their exploitation as an adjuvant and a delivery system, and address important areas of future research interest.

Synergistic effect of high-mobility group box-1 and lipopolysaccharide on cytokine induction in bovine peripheral blood mononuclear cells.

High-mobility group box 1 (HMGB1) is one of the potent endogenous adjuvants released by necrotic and activated innate immune cells. HMGB1 modulates innate and adaptive immune responses in humans and mice by mediating immune cells crosstalk. However, the immuno-modulatory effects of HMGB1 in the bovine immune system are not clearly known. In this study, the effect of bovine HMGB1 alone or in combination with LPS on the expression kinetics of cytokines upon in vitro stimulation of bovine peripheral blood mononuclear cells (PBMCs) was investigated by quantitative PCR assay. The biological activity of bovine HMGB1 expressed in this prokaryotic expression system was confirmed by its ability to induce nitric oxide secretion in RAW 264.7 cells. The present results indicate that HMGB1 induces a more delayed TNF-α response than does LPS in stimulated PBMCs. However, IFN-γ, IFN-ß and IL-12 mRNA transcription peaked at 6 hr post stimulation after both treatments. Further, HMGB1 and LPS heterocomplex up-regulated TNF-α, IFN-γ and IL-12 mRNA expression significantly than did individual TLR4 agonists. The heterocomplex also enhanced the expression of TLR4 on bovine PBMCs. In conclusion, the data indicate that HMGB1 and LPS act synergistically and enhance proinflammatory cytokines, thereby eliciting Th1 responses in bovine PBMCs. These results suggest that HMGB1 can act as an adjuvant in modulating the bovine immune system and thus lays a foundation for using HMGB1 as an adjuvant in various bovine vaccine preparations.

Kinetics of cytokine expression in bovine PBMCs and whole blood after in vitro stimulation with foot-and-mouth disease virus (FMDV) antigen.

The interest in analysing antigen-specific cytokine responses has substantially increased in recent years, in part due to their use in assessing vaccine efficacy. In the present study, the kinetics of IL-2, IL-4 and IFN-γ expression was determined in bovine PBMCs by real-time PCR and in whole blood by cytokine-release assay after in vitro stimulation with recall foot-and-mouth disease virus (FMDV) antigen. The results showed that the cytokine mRNA of IL-2 and IFN-γ in PBMCs were induced early (peak induction at 6 h), whereas the IL-4 mRNA showed delayed induction (peaked at 24 h). In contrast, the kinetics of cytokine proteins in whole blood was different and required the accumulation of the proteins before being optimally detected. The peak accumulation of cytokine protein in whole blood was recorded at 72 h for IL-2 and IL-4, and 96 h for IFN-γ. The findings of this study are of importance when selecting an optimal time points for measuring antigen-specific cytokine expression in cattle.

Immunological evaluation of mannosylated chitosan nanoparticles based foot and mouth disease virus DNA vaccine, pVAC FMDV VP1-OmpA in guinea pigs.

A DNA vaccine for foot and mouth disease (FMD) based on mannosylated chitosan nanoparticles was evaluated in guinea pigs. The DNA construct was comprised of FMD virus full length-VP1 gene and outer membrane protein A (Omp A) gene of Salmonella typhimurium as a Toll-like receptor (TLR)-ligand in pVAC vector. Groups of guinea pigs immunized either intramuscularly or intra-nasally were evaluated for induction of virus neutralizing antibodies, Th1(IgG2) and Th2 (IgG1) responses, lymphocyte proliferation, reactive nitrogen intermediate production, secretory IgA for naso-mucosal immune response and protection upon homotypic type O virulent FMD virus challenge. The results indicate the synergistic effect of OmpA on the immunogenic potential of FMD DNA vaccine construct delivered using mannosylated chitosan nano-particles by different routes of administration. These observations suggest the substantial improvement in all the immunological parameters with enhanced protection in guinea pigs.

Linking S. aureus Immune Evasion Mechanisms to Staphylococcal Vaccine Failures.

Vaccination arguably remains the only long-term strategy to limit the spread of S. aureus infections and its related antibiotic resistance. To date, however, all staphylococcal vaccines tested in clinical trials have failed. In this review, we propose that the failure of S. aureus vaccines is intricately linked to prior host exposure to S. aureus and the pathogen's capacity to evade adaptive immune defenses. We suggest that non-protective immune imprints created by previous exposure to S. aureus are preferentially recalled by SA vaccines, and IL-10 induced by S. aureus plays a unique role in shaping these non-protective anti-staphylococcal immune responses. We discuss how S. aureus modifies the host immune landscape, which thereby necessitates alternative approaches to develop successful staphylococcal vaccines.

The characteristics of pre-existing humoral imprint determine efficacy of S. aureus vaccines and support alternative vaccine approaches.

The failure of the Staphylococcus aureus (SA) IsdB vaccine trial can be explained by the recall of non-protective immune imprints from prior SA exposure. Here, we investigate natural human SA humoral imprints to understand their broader impact on SA immunizations. We show that antibody responses against SA cell-wall-associated antigens (CWAs) are non-opsonic, while antibodies against SA toxins are neutralizing. Importantly, the protective characteristics of the antibody imprints accurately predict the failure of corresponding vaccines against CWAs and support vaccination against toxins. In passive immunization platforms, natural anti-SA human antibodies reduce the efficacy of the human monoclonal antibodies suvratoxumab and tefibazumab, consistent with the results of their respective clinical trials. Strikingly, in the absence of specific humoral memory responses, active immunizations are efficacious in both naive and SA-experienced mice. Overall, our study points to a practical and predictive approach to evaluate and develop SA vaccines based on pre-existing humoral imprint characteristics.

Functional divergence of a bacterial enzyme promotes healthy or acneic skin.

Acne is a dermatologic disease with a strong pathologic association with human commensal Cutibacterium acnes. Conspicuously, certain C. acnes phylotypes are associated with acne, whereas others are associated with healthy skin. Here we investigate if the evolution of a C. acnes enzyme contributes to health or acne. Two hyaluronidase variants exclusively expressed by C. acnes strains, HylA and HylB, demonstrate remarkable clinical correlation with acne or health. We show that HylA is strongly pro-inflammatory, and HylB is modestly anti-inflammatory in a murine (female) acne model. Structural and phylogenic studies suggest that the enzymes evolved from a common hyaluronidase that acquired distinct enzymatic activity. Health-associated HylB degrades hyaluronic acid (HA) exclusively to HA disaccharides leading to reduced inflammation, whereas HylA generates large-sized HA fragments that drive robust TLR2-dependent pathology. Replacing an amino acid, Serine to Glycine near the HylA catalytic site enhances the enzymatic activity of HylA and produces an HA degradation pattern intermediate to HylA and HylB. Selective targeting of HylA using peptide vaccine or inhibitors alleviates acne pathology. We suggest that the functional divergence of HylA and HylB is a major driving force behind C. acnes health- and acne- phenotype and propose targeting of HylA as an approach for acne therapy.

Comparative Transcriptome Analysis of Acne vulgaris, Rosacea, and Hidradenitis Suppurativa Supports High Dose Dietary Zinc as a Therapeutic Agent.

Acne vulgaris, rosacea, and hidradenitis suppurativa are enduring inflammatory skin conditions that frequently manifest with akin clinical attributes, posing a considerable challenge for their distinctive diagnosis. While these conditions do exhibit certain resemblances, they also demonstrate distinct underlying pathophysiological mechanisms and treatment modalities. Delving into both the molecular parallels and disparities among these three disorders can yield invaluable insights for refined diagnostics, effective management, and targeted therapeutic interventions. In this report, we present a comparative analysis of transcriptomic data across these three diseases, elucidating differentially expressed genes and enriched pathways specific to each ailment, as well as those shared among them. We also identified high dose dietary zinc as a potential therapeutic agent and validated its efficacy in an acne mouse model.

Integrating complex host-pathogen immune environments into S. aureus vaccine studies.

Staphylococcus aureus (SA) is a leading cause of bacterial infection and antibiotic resistance globally. Therefore, development of an effective vaccine has been a major goal of the SA field for the past decades. With the wealth of understanding of pathogenesis, the failure of all SA vaccine trials has been a surprise. We argue that experimental SA vaccines have not worked because vaccines have been studied in naive laboratory animals, whereas clinical vaccine efficacy is tested in immune environments reprogrammed by SA. Here, we review the failed SA vaccines that have seemingly defied all principles of vaccinology. We describe major SA evasion strategies and suggest that they reshape the immune environment in a way that makes vaccines prone to failures. We propose that appropriate integration of concepts of host-pathogen interaction into vaccine study designs could lead to insight critical for the development of an effective SA vaccine.

Non-protective immune imprint underlies failure of Staphylococcus aureus IsdB vaccine.

Humans frequently encounter Staphylococcus aureus (SA) throughout life. Animal studies have yielded SA candidate vaccines, yet all human SA vaccine trials have failed. One notable vaccine "failure" targeted IsdB, critical for host iron acquisition. We explored a fundamental difference between humans and laboratory animals-natural SA exposure. Recapitulating the failed phase III IsdB vaccine trial, mice previously infected with SA do not mount protective antibody responses to vaccination, unlike naive animals. Non-protective antibodies exhibit increased α2,3 sialylation that blunts opsonophagocytosis and preferentially targets a non-protective IsdB domain. IsdB vaccination of SA-infected mice recalls non-neutralizing humoral responses, further reducing vaccine efficacy through direct antibody competition. IsdB vaccine interference was overcome by immunization against the IsdB heme-binding domain. Purified human IsdB-specific antibodies also blunt IsdB passive immunization, and additional SA vaccines are susceptible to SA pre-exposure. Thus, failed anti-SA immunization trials could be explained by non-protective imprint from prior host-SA interaction.

Anti-Inflammatory Properties of Plasma from Children with Short Bowel Syndrome.

Sepsis, resulting from a dysregulated host immune response to invading pathogens, is the leading cause of mortality in critically ill patients worldwide. Immunomodulatory treatment for sepsis is currently lacking. Children with short bowel syndrome (SBS) may present with less severe symptoms during gram-negative bacteremia. We, therefore, tested the hypothesis that plasma from children with SBS could confer protection against Escherichia coli sepsis. We showed that SBS plasma at 5% and 10% concentrations significantly (p < 0.05) inhibited the production of both TNF-α and IL-6 induced by either E. coli- or LPS-stimulated host cells when compared to plasma from healthy controls. Furthermore, mice treated intravenously with select plasma samples from SBS or healthy subjects had reduced proinflammatory cytokine levels in plasma and a significant survival advantage after E. coli infection. However, SBS plasma was not more protective than the plasma of healthy subjects, suggesting that children with SBS have other immunomodulatory mechanisms, in addition to neutralizing antibodies, to alleviate their symptoms during gram-negative sepsis.

Intranasally administered polyethylenimine adjuvanted influenza M2 ectodomain induces partial protection against H9N2 influenza A virus infection in chickens.

This study aimed to investigate whether intranasally coadministered four tandem copies of extracellular domains of M2 (M2e) and polyethyleneimine (PEI), a mucosal adjuvant, can protect chickens against H9N2 influenza A virus infection. Groups of chickens were intranasally vaccinated with M2e plus PEI adjuvant, M2e alone or PEI adjuvant, and antibody (serum IgG and mucosal IgA) and cellular (CD4+ T cells and IFN-γ levels) immune responses were measured post-vaccination. We demonstrated that the chickens vaccinated with M2e plus PEI adjuvant showed significantly (p < 0.05) higher M2e-specific systemic IgG and mucosal IgA responses compared to the chickens that received either M2e alone or PEI adjuvant. The IgA responses measured in lungs were almost comparable to that of the serum IgG levels. Upon restimulation of the vaccinated peripheral blood mononuclear cells (PBMCs) with M2e antigen, significantly (p < 0.05) higher IFN-γ levels were observed only in M2e plus PEI adjuvant vaccinated group. Lymphoproliferative and CD4+ T cell responses, as measured by MTT-based assay and flow cytometry, respectively, were also observed significantly (p < 0.05) higher in M2e plus PEI adjuvant vaccinated chickens. On challenge with the H9N2 virus (104TCID50) at 28th day post-vaccination, M2e plus PEI adjuvant vaccinated group exhibited lower lung inflammation and viral load compared to the chickens treated with either M2e alone or PEI adjuvant. In summary, we show that intranasally coadministered M2e and PEI adjuvant can elicit humoral and cell-mediated immune responses and can reduce viremia levels in chickens post H9N2 infection in chickens.

Human antigen presenting cells stimulated with Salmonella delivered influenza antigens induce cytokine production and proliferation of human CD4+ T cells in vitro.

This study aimed to investigate whether the human antigen presenting cells (APCs) can process and present Salmonella expressing H7N9 hemagglutinin (Sal-HA), neuraminidase (Sal-NA) or M2 ectodomain (Sal-M2e) to T cells and subsequently activate CD4+ T cell responses in vitro. In this study, APCs generated from human peripheral blood mononuclear cells (PBMCs) were first treated with mitomycin-C, followed by stimulation with Sal-HA, Sal-M2e, Sal-NA or Salmonella alone for 24 h. Subsequently, stimulated APCs were coincubated with untreated PBMCs (1:10) of the same individual for 24 or 72 h and then analysed for cytokine induction and T cell proliferations by qRT-PCR assay and flow cytometry, respectively. Our results demonstrated that APCs stimulated with Sal-HA, Sal-M2e or Sal-NA induced significantly (p < .05) higher CD3+CD4+ T cell proliferations compared to the APCs treated with Salmonella alone. Our data further revealved that APCs treated with Sal-HA induced significantly (p < .05) higher CD3+CD4+ T cell responses compared to the APCs treated with either Sal-M2e or Sal-NA, which both induced almost comparable levels. The T cell proliferation responses were further measured by lymphocyte proliferation assay and the results showed that Sal-HA and Sal-M2e stimulated APCs induced significantly (p < .05) higher proliferations in T cells compared to the APCs stimulated with either Sal-NA or Salmonella alone. With respect to cytokine inductions, APCs treated with either Sal-HA or Sal-M2e induced significantly (p < .05) higher mRNA transcription levels of proinflammatory (IL-1ß, IL-6, IL-12 and IL-23), Th1 (IFN-γ), Th17 (IL-17 and IL-21) and Th2 (IL-10 and TGF-ß) cytokines in T cells compared to Sal-NA or Salmonella alone treated APCs. In conclusion, we show that Salmonella system can efficiently deliver vaccine antigens to APCs and is, thus, capable to elicit heterologous antigen-specific adaptive immunity.