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.

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.

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.

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.

Intranasally administered anti-Brucella subunit vaccine formulation induces protective immune responses against nasal Brucella challenge.

The present study was aimed to develop a safe and effective anti-Brucella subunit vaccine for mucosal protection against the respiratory exposure of Brucella infection. A chitosan-based Brucella nasal vaccine (BNV) was formulated using well-known Brucella immunogens, sodC, omp19, BLS and PrpA and tested against nasal Brucella challenge in BALB/c mice. The mice were intra-nasally vaccinated with sterile phosphate buffer saline (PBS), BNV or BNV plus Brucella LPS, and humoral (systemic IgG and mucosal IgA) and cell-mediated immune responses were analyzed. Results showed that mice vaccinated with either BNV or BNV plus LPS elicited significantly (p < 0.05) high IgG and IgA responses compared to the PBS control. The IgG responses were significantly (p < 0.05) higher than IgA levels, which showed almost comparable levels observed in either intestines or in lungs. Furthermore, the IgG and IgA responses against each individual component of the BNV formulation indicated that omp19 induced highest levels of both IgG and IgA levels than the other constituents of BNV formulation. Upon re-stimulation of the splenocytes with Brucella whole cell lysate, significantly (p < 0.05) high IFN-γ levels, lymphocyte proliferation, and CD4+ T cell responses were observed in mice vaccinated with BNV or BNV plus LPS. Upon sub-lethal nasal challenge with wild-type Brucella strain, vaccinated mice showed significant reduction of Brucella recovery in lungs and spleen compared to the PBS control. This study indicates that BNV formulation with or without Brucella LPS efficiently induced humoral and cell-mediated immune responses and conferred significant protection against the sub-lethal Brucella challenge.

An Influenza HA and M2e Based Vaccine Delivered by a Novel Attenuated Salmonella Mutant Protects Mice against Homologous H1N1 Infection.

Attenuated Salmonella strains constitute a promising technology for the development of a more efficient multivalent protein based vaccines. In this study, we constructed a novel attenuated strain of Salmonella for the delivery and expression of the H1N1 hemagglutinin (HA) and the conserved extracellular domain of the matrix protein 2 (M2e). We demonstrated that the constructed Salmonella strain exhibited efficient HA and M2e protein expressions and little cytotoxicity and pathogenicity in mice. Using BALB/c mice as the model, we showed that the mice vaccinated with a Salmonella strain expressing HA and M2e protein antigens, respectively, induced significant production of HA and M2e-specific serum IgG1 and IgG2a responses, and of anti-HA interferon-γ producing T cells. Furthermore, immunization with Salmonella-HA-M2e-based vaccine via different routes provided protection in 66.66% orally, 100% intramuscularly, and 100% intraperitoneally immunized mice against the homologous H1N1 virus while none of the animals survived treated with either the PBS or the Salmonella carrying empty expression vector. Ex vivo stimulated dendritic cells (DCs) with heat killed Salmonella expressing HA demonstrated that DCs play an important role in the elicitation of HA-specific humoral immune responses in mice. In summary, Salmonella-HA-M2e-based vaccine elicits efficient antigen-specific humoral and cellular immune responses, and provides significant immune protection against a highly pathogenic H1N1 influenza virus.

Bacterial flagellin-a potent immunomodulatory agent.

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.

Co-administration of flagellin augments immune responses to inactivated foot-and-mouth disease virus (FMDV) antigen.

Foot-and-mouth disease virus (FMDV) is one of the most contagious animal virus known that affects livestock health and production. This study aimed to investigate the effect of flagellin, a toll-like receptor 5 agonist, on the immune responses to inactivated FMDV antigen in guinea pig model. Our results showed that the co-administration of flagellin with FMDV antigen through intradermal route induces earlier and higher anti-FMDV neutralizing antibody responses as compared to FMDV antigen alone. Both IgG1 and IgG2 antibody-isotype responses were enhanced, but the IgG1/IgG2 ratios were relatively low, indicative of TH1 type of immune activation. On live viral challenge, flagellin+FMDV immunized guinea pigs showed 70% (7 out of 10) protection rate as compared to 40% (4 out of 10) in FMDV alone immunized guinea pigs. The results demonstrate that the co-administration of flagellin augments immune responses (preferably TH1 type) and protective efficacy against FMDV in guinea pigs.