Suppression of interleukin-33 bioactivity through proteolysis by apoptotic caspases.

Interleukin-33 (IL-33) is a member of the IL-1 family and is involved in polarization of T cells toward a T helper 2 (Th2) cell phenotype. IL-33 is thought to be activated via caspase-1-dependent proteolysis, similar to the proinflammatory cytokines IL-1 beta and IL-18, but this remains unproven. Here we showed that IL-33 was processed by caspases activated during apoptosis (caspase-3 and -7) but was not a physiological substrate for caspases associated with inflammation (caspase-1, -4, and -5). Furthermore, caspase-dependent processing of IL-33 was not required for ST2 receptor binding or ST2-dependent activation of the NF-kappaB transcription factor. Indeed, caspase-dependent proteolysis of IL-33 dramatically attenuated IL-33 bioactivity in vitro and in vivo. These data suggest that IL-33 does not require proteolysis for activation, but rather, that IL-33 bioactivity is diminished through caspase-dependent proteolysis within apoptotic cells. Thus, caspase-mediated proteolysis acts as a switch to dampen the proinflammatory properties of IL-33.

Targeted nasal vaccination provides antibody-independent protection against Staphylococcus aureus.

Despite showing promise in preclinical models, anti-Staphylococcus aureus vaccines have failed in clinical trials. To date, approaches have focused on neutralizing/opsonizing antibodies; however, vaccines exclusively inducing cellular immunity have not been studied to formally test whether a cellular-only response can protect against infection. We demonstrate that nasal vaccination with targeted nanoparticles loaded with Staphylococcus aureus antigen protects against acute systemic S. aureus infection in the absence of any antigen-specific antibodies. These findings can help inform future developments in staphylococcal vaccine development and studies into the requirements for protective immunity against S. aureus.

miRNAs as biomarkers of therapeutic response to HER2-targeted treatment in breast cancer: A systematic review.

Breast cancer is the most common type of lethal cancer in women globally. Women have a 1 in 8 chance of developing breast cancer in their lifetime. Among the four primary molecular subtypes (luminal A, luminal B, HER2+, and triple-negative), HER2+ accounts for 20-25 % of all breast cancer and is rather aggressive. Although the treatment outcome of HER2+ breast cancer patients has been significantly improved with anti-HER2 agents, primary and acquired drug resistance present substantial clinical issues, limiting the benefits of HER2-targeted treatment. MicroRNAs (miRNAs) play a central role in regulating acquired drug resistance. miRNA are single-stranded, non-coding RNAs of around 20-25 nucleotides, known for essential roles in regulating gene expression at the post-transcriptional level. Increasing evidence has demonstrated that miRNA-mediated alteration of gene expression is associated with tumorigenesis, metastasis, and tumor response to treatment. Comprehensive knowledge of miRNAs as potential markers of drug response can help provide valuable guidance for treatment prognosis and personalized medicine for breast cancer patients.

The vaccine adjuvant alum inhibits IL-12 by promoting PI3 kinase signaling while chitosan does not inhibit IL-12 and enhances Th1 and Th17 responses.

Alum is the principal vaccine adjuvant for clinical applications but it is a poor inducer of cellular immunity and is not an optimal adjuvant for vaccines where Th1 responses are required for protection. The mechanism underlying the inefficiency of alum in promoting Th1 responses is not fully understood. We show that aluminium hydroxide, aluminium phosphate, and calcium phosphate adjuvants inhibit the secretion of the Th1 polarizing cytokine, IL-12 by dendritic cells (DCs). Alum selectively inhibited DC expression of the IL-12p35 subunit and the inhibitory effect results from adjuvant-induced PI3 kinase signaling. To develop a more effective adjuvant for promoting cell-mediated immunity, we investigated alternative particulates and found that in contrast to alum, the cationic polysaccharide chitosan did not inhibit IL-12 secretion. A combination of chitosan and the TLR9 agonist CpG activated the NLRP3 inflammasome and enhanced secretion of IL-12 and the other key Th1 and Th17-cell polarizing cytokines. When used as an adjuvant, CpG-chitosan induced NLRP3-dependent antigen-specific Th1 and Th17 responses. A combination of alum and CpG also enhanced Th1 and Th17 responses but was less effective than CpG-chitosan. Therefore, chitosan is an attractive alternative to alum in adjuvants for vaccines where potent cell-mediated immunity is required.

Pneumolysin activates the NLRP3 inflammasome and promotes proinflammatory cytokines independently of TLR4.

Pneumolysin (PLY) is a key Streptococcus pneumoniae virulence factor and potential candidate for inclusion in pneumococcal subunit vaccines. Dendritic cells (DC) play a key role in the initiation and instruction of adaptive immunity, but the effects of PLY on DC have not been widely investigated. Endotoxin-free PLY enhanced costimulatory molecule expression on DC but did not induce cytokine secretion. These effects have functional significance as adoptive transfer of DC exposed to PLY and antigen resulted in stronger antigen-specific T cell proliferation than transfer of DC exposed to antigen alone. PLY synergized with TLR agonists to enhance secretion of the proinflammatory cytokines IL-12, IL-23, IL-6, IL-1ß, IL-1α and TNF-α by DC and enhanced cytokines including IL-17A and IFN-γ by splenocytes. PLY-induced DC maturation and cytokine secretion by DC and splenocytes was TLR4-independent. Both IL-17A and IFN-γ are required for protective immunity to pneumococcal infection and intranasal infection of mice with PLY-deficient pneumococci induced significantly less IFN-γ and IL-17A in the lungs compared to infection with wild-type bacteria. IL-1ß plays a key role in promoting IL-17A and was previously shown to mediate protection against pneumococcal infection. The enhancement of IL-1ß secretion by whole live S. pneumoniae and by PLY in DC required NLRP3, identifying PLY as a novel NLRP3 inflammasome activator. Furthermore, NLRP3 was required for protective immunity against respiratory infection with S. pneumoniae. These results add significantly to our understanding of the interactions between PLY and the immune system.

A novel adjuvanted capsule based strategy for oral vaccination against infectious diarrhoeal pathogens.

Diarrhoeal infections are a major cause of morbidity and mortality with enterotoxigenic Escherichia coli (ETEC) and cholera imposing a significant global burden. There is currently no licensed vaccine for ETEC. Development of new nonliving oral vaccines has proven difficult due to the physicochemical and immunological challenges associated with the oral route. This demands innovative delivery solutions to protect antigens, control their release and build in immune-stimulatory activity. We describe the Single Multiple Pill® (SmPill®) vaccine formulation which combines the benefits of enteric polymer coating to protect against low gastric pH, a dispersed phase to control release and aid the solubility of non-polar components and an optimized combination of adjuvant and antigen to promote mucosal immunity. We demonstrate the effectiveness of this system with whole cell killed E. coli overexpressing colonization factor antigen I (CFA/I), JT-49. Alpha-galactosylceramide was identified as a potent adjuvant within SmPill® that enhanced the immunogenicity of JT-49. The bacteria associated with the dispersed phase were retained within the capsules at gastric pH but released at intestinal pH. Vaccination with an optimized SmPill® formulation promoted CFA/I-specific immunoglobulin A (IgA) responses in the intestinal mucosa in addition to serum IgG and a solubilized adjuvant was indispensable for efficacy.

Induction of protective serum meningococcal bactericidal and diphtheria-neutralizing antibodies and mucosal immunoglobulin A in volunteers by nasal insufflations of the Neisseria meningitidis serogroup C polysaccharide-CRM197 conjugate vaccine mixed with chitosan.

Thirty-six healthy volunteers received either a single intramuscular injection of Neisseria meningitidis serogroup C polysaccharide (MCP)-CRM197 conjugate vaccine in alum or two nasal insufflations 28 days apart of the same vaccine powder, without alum, mixed with chitosan. Nasal immunization was well tolerated, with fewer symptoms reported than after intramuscular injection. The geometric mean concentrations of MCP-specific immunoglobulin G (IgG) after one nasal immunization were 3.25 microg/ml in naïve subjects and 14.4 microg/ml in subjects previously immunized parenterally, compared with 4.30 microg/ml in naïve subjects immunized intramuscularly. The geometric mean titer of serum bactericidal antibody (SBA) rose 24-fold after two nasal immunizations in naïve subjects and was comparable to parenteral immunization (1,080 versus 1,625). All subjects achieved SBA titers associated with protection after two nasal immunizations: even those with titers of <8 at entry. A single nasal immunization boosted the SBA titer to > or =128 in 96% of previously immunized subjects, and two immunizations achieved this level in 92% of naive subjects. MCP-specific IgG levels were approximately 70% IgG2 and approximately 20% IgG1 after nasal or intramuscular immunization. Increases in CRM197-specific IgG and diphtheria toxin-neutralizing activity were observed after nasal or intramuscular immunization, with balanced IgG1/IgG2 and higher IgG4. Significant MCP-specific secretory IgA was detected in nasal wash only after nasal immunization and predominantly on the immunized side. Simple nasal insufflation of existing MCP-CRM197 conjugate vaccines in chitosan offers an inexpensive but effective needle-free prime and boost against serogroup C N. meningitidis and diphtheria.

Intranasal immunization with genetically detoxified diphtheria toxin induces T cell responses in humans: enhancement of Th2 responses and toxin-neutralizing antibodies by formulation with chitosan.

We previously reported that intranasal immunization with a non-toxic mutant cross-reacting material (CRM)197 of diphtheria toxin, formulated with chitosan, generated protective neutralizing antibodies in mice and guinea pigs. Furthermore, we demonstrated that intranasal delivery of a powder formulation of the CRM197-based vaccine was well tolerated and significantly boosted antibody responses in adult volunteers. Here we report that intranasal booster immunization with CRM197 alone or with chitosan induced systemic T cell responses. We addressed for the first time the induction of T cell subtypes following intranasal vaccination in humans. Intranasal vaccination with CRM197, like parenteral immunization with a conventional diphtheria toxoid vaccine, enhanced antigen-specific IFN-gamma production. However, formulation of the nasal diphtheria vaccine with chitosan significantly augmented Th2-type responses, which correlated with protective levels of toxin-neutralizing antibodies in intranasally boosted individuals. The results suggest that vaccines capable of inducing strong Th2-type responses, such as CRM197 formulated with chitosan, have potential for the development of a protective mucosal vaccine against diphtheria in humans. Furthermore, our findings demonstrate that mucosal subunit vaccines with appropriate delivery systems have considerable potential for booster immunization of adults.

Protective levels of diphtheria-neutralizing antibody induced in healthy volunteers by unilateral priming-boosting intranasal immunization associated with restricted ipsilateral mucosal secretory immunoglobulin a.

Subunit intranasal vaccines offer the prospect of inducing combined systemic-mucosal immunity against mucosally transmitted infections such as human immunodeficiency virus. However, although human studies have demonstrated the induction of active immunity, secretory immunoglobulin A (sIgA) responses are variable, and no study has demonstrated protection by accepted vaccine-licensing criteria as measured by direct toxin-neutralizing activity. Using the genetically inactivated mutant diphtheria toxoid CRM(197) in a bioadhesive polycationic polysaccharide chitosan delivery system, we found that a single nasal immunization was well tolerated and boosted antitoxin neutralizing activity in healthy volunteers, which could be further boosted by a second immunization. The neutralizing activity far exceeded accepted protective levels and was equivalent to that induced by standard intramuscular vaccine and significantly greater than intranasal immunization with CRM(197) in the absence of chitosan. A striking but unexpected observation was that although unilateral intranasal immunization induced circulating antitoxin antibody-secreting cells, a nasal antitoxin sIgA response was seen only after the second immunization and only in the vaccinated nostril. If these data are reproduced in larger studies, an intranasal diphtheria vaccine based on CRM(197)-chitosan could be rapidly licensed for human use. However, a restricted sIgA response suggests that care must be taken in the priming-boosting strategy and clinical sampling techniques when evaluating such vaccines for the induction of local mucosal immunity.

Mucosal vaccination against serogroup B meningococci: induction of bactericidal antibodies and cellular immunity following intranasal immunization with NadA of Neisseria meningitidis and mutants of Escherichia coli heat-labile enterotoxin.

Conjugated polysaccharide vaccines protect against serogroup C meningococci. However, this approach cannot be applied to serogroup B, which is still a major cause of meningitis. We evaluated the immunogenicity of three surface-exposed proteins from serogroup B Neisseria meningitidis (App, NhhA, and NadA) identified during whole-genome sequencing. Mice were immunized intranasally with individual proteins in the presence of wild-type Escherichia coli heat-labile enterotoxin (LTwt), LTR72, a partially inactivated mutant, or LTK63, a completely nontoxic mutant, as the adjuvant. Each of the meningococcal proteins induced significant cellular responses; NhhA and NadA induced strong antibody responses, but only NadA induced bactericidal antibody when administered intranasally with mucosal adjuvants. In addition, immunoglobulin A and bactericidal antibodies were detected in the respiratory tract following intranasal delivery of NadA. Analysis of antigen-specific cytokine production by T cells from immunized mice revealed that intranasal immunization with NadA alone failed to generate detectable cellular immune responses. In contrast, LTK63, LTR72, and LTwt significantly augmented NadA-specific gamma interferon, interleukin-4 (IL-4), IL-5, and IL-10 production by spleen and lymph node cells, suggesting that both Th1 and Th2 cells were induced in vivo. The strongest cellular responses and highest bactericidal antibody titers were generated with LTR72 as the adjuvant. These findings demonstrate that the quality and magnitude of the immune responses generated by mucosal vaccines are influenced by the antigen as well as the adjuvant and suggest that nasal delivery of NadA with mucosal adjuvants has considerable potential in the development of a mucosal vaccine against serogroup B meningococci.