Antimicrobial peptides in mucosal secretions: the importance of local secretions in mitigating infection

The antimicrobial activity of the collective molecules comprising human milk reflects an evolutionarily successful paradigm for preventing and limiting microbial infection. Understanding the molecules that participate in this process and how they work can yield insight into potentially new antimicrobial therapies. Upon proteolytic processing, antimicrobial peptides can be derived from milk proteins, such as lactoferrin, casein, and lysozyme. Similarly, using the HIV-1 gp41 protein template, we have demonstrated that the 28-residue C-terminus, when produced as an independent peptide, exhibits selective toxicity for bacteria over eukaryotic cells. Upon optimizing this sequence for cationic charge and hydrophobic character presented as a alpha-helical structure, we show improved capability of the parent LLP1 sequence to selectively kill bacteria in the host environment and that this activity is increased by the inclusion of Trp residues on the hydrophobic face. We report that it is possible to (i) design de novo antimicrobial peptides that demonstrate optimal antimicrobial activity with minimal inflammatory activity and (ii) design antimicrobial peptides to function in a defined environment. In the end, we describe a de novo designed antimicrobial peptide, WLBU2, which is selectively toxic to microbial pathogens in complex environments and does not stimulate a significant immunomodulatory response. In spite of these properties, WLBU2 activity against Pseudomonas aeruginosa in human milk is inferior to the host peptide LL37 with regard to antimicrobial potency. These studies demonstrate that antimicrobial peptides can be engineered for greater potency in one medium but may not be optimal for working in a different medium such as human milk.     

Milk IgA responses are augmented by antigen delivery to the mucosal addressin cellular adhesion molecule 1

The mucosal addressin cellular adhesion molecule 1 (MAdCAM) is expressed on the venules of the gut associated lymphoid tissue (GALT); it is also expressed on the venules of the lobules of the mammary gland. We have previously found that MAdCAM-targeting using a rat anti-MAdCAM monoclonal Ab as both antigen and targeting moiety resulted in an enhanced local IgA gut response. We therefore surmised that such targeting may also enhance IgA responses in the mammary gland. We show that our model antigen localizes to the lobules of the mammary glands as well as the GALT, but not to the draining lymph nodes and that targeting MAdCAM results in secretory IgA responses in the milk. We provide evidence that this milk IgA Ab is of a secretory nature and is consistent with derivation from gut plasmablasts that have migrated to the mammary gland. Targeting MAdCAM may be a way for a novel vaccine strategy that affords protection to the mammary gland and the suckling neonate.     

Oral retinyl palmitate or retinoic acid corrects mucosal IgA responses toward an intranasal influenza virus vaccine in vitamin A deficient mice

Vitamin A deficiency (VAD) is a leading cause of pediatric morbidity and mortality due to infectious diseases. Recent pre-clinical studies have revealed that VAD impairs mucosal IgA-producing antibody forming cell (AFC) responses toward a paramyxovirus vaccine in the upper respiratory tract (URT), thus impeding a first line of defense at the pathogen's point-of-entry. The studies described here tested the hypothesis that VAD may also impair immune responses after FluMist vaccinations. Results show that (i) IgA-producing antibody forming cells (AFCs) are significantly reduced following FluMist vaccination in VAD mice, and (ii) oral doses of either retinyl palmitate or retinoic acid administered on days 0, 3, and 7 relative to vaccination rescue the response. Data encourage the conduct of clinical studies to determine if there are FluMist vaccine weaknesses in human VAD populations and to test corrective supplementation strategies. Improvements in vaccine efficacy may ultimately reduce the morbidity and mortality caused by influenza virus worldwide.     

Induction of serum and mucosal FIV-specific immune responses by intranasal immunization with p24Gag

We examined the ability of FIV p24Gag to induce systemic and mucosal FIV-specific immune responses when delivered as a nasal immunogen alone, or with a mucosal adjuvant, Escherichia coli heat labile toxin LT(R192G). Nasal immunization with p24Gag alone induced FIV-specific immune responses but overall responses were weak, transient, and/or present only in a few animals. Co-administration of LT(R192G) resulted in strong FIV-specific serum IgG and enhanced salivary IgA responses. Moreover, FIV-specific IgA was detected in vaginal wash fluid from 6/6 cats co-immunized with LT(R192G) and p24Gag versus 1/6 immunized with p24Gag alone. This is the first report detailing induction of systemic or mucosal FIV-specific immune responses by nasal immunization alone. As such, this study demonstrates that nasal immunization of cats can be a relevant and effective route for the delivery of candidate vaccines. However, while nasal immunization of cats with p24Gag induces antigen-specific systemic immune responses, development of strong systemic and mucosal immune responses requires co-administration of a mucosal adjuvant, such as LT(R192G).     

Nanoparticle size influences the magnitude and quality of mucosal immune responses after intranasal immunization

Background

The development of nanoparticulate antigen-delivery systems is an important emerging area of vaccinology, being sought to amplify immune responses to recombinant antigens that are poorly immunogenic. Nanoparticle size may play an important role in influencing the activity of such particulate-based adjuvants.

Methods

To explore how the size of nanoparticles that are in the range of many common viruses can modulate the magnitude and quality of mucosal immune responses, the model antigen ovalbumin (OVA) was conjugated to 30 nm or 200 nm polypropylene sulfide nanoparticles (NPs) and administered intranasally to C57BL/6 mice.

Results

We show that by increasing the size of the NPs from 30 to 200 nm, OVA was more effectively delivered into both MHC class I and MHC class II-presentation pathways. Intranasal immunization with the 200 nm NPs increased the magnitude of CD4⁺ T cell responses in the lungs, as well as systemic and mucosal humoral responses. Most importantly, 200 nm NPs increased the proportion of antigen-specific polyfunctional CD4⁺ T cells as compared to 30 nm NPs.

Conclusions

The 200 nm NPs are a very interesting antigen nanocarrier for prophylactic vaccines against mucosal pathogens that require multifunctional CD4⁺ T cells for protection. These results contribute to our understanding of how the size of an antigen-conjugated nanoparticle modulates mucosal immune responses to a protein antigen and may be useful to engineer subunit vaccines able to elicit appropriate mucosal immune responses that correlate with protection.     

Immunopotentiating of mucosal and systemic antibody responses in mice by intranasal immunization with HLT-combined influenza virosomal vaccine

The mucosal vaccination strategy against influenza has been investigated by using influenza virosomal vaccine (IRIV) combined with two different adjuvants: the procholeragenoid (PCG) and the Escherichia coli heat labile toxin (HLT). A comparative study has been carried out on mice administered intranasally with these different formulations of influenza vaccine. PCG appears less effective than HLT in inducing an IgG response, but both the adjuvants elicit mucosal adjuvant activity inducing s-IgA in the upper respiratory tract. On the contrary, only HLT when administered intranasally to mice with influenza virosomes stimulates the production of s-IgA in the lower respiratory tract thereby providing a better protection against primary infection of the respiratory system. Both HLT and PCG enhance the production of IFN-gamma in the respiratory tract, nevertheless HLT appears more efficacious as a mucosal adjuvant.     

Carnauba wax nanoparticles enhance strong systemic and mucosal cellular and humoral immune responses to HIV-gp140 antigen

Induction of humoral responses to HIV at mucosal compartments without inflammation is important for vaccine design. We developed charged wax nanoparticles that efficiently adsorb protein antigens and are internalized by DC in the absence of inflammation. HIV-gp140-adsorbed nanoparticles induced stronger in vitro T-cell proliferation responses than antigen alone. Such responses were greatly enhanced when antigen was co-adsorbed with TLR ligands. Immunogenicity studies in mice showed that intradermal vaccination with HIV-gp140 antigen-adsorbed nanoparticles induced high levels of specific IgG. Importantly, intranasal immunization with HIV-gp140-adsorbed nanoparticles greatly enhanced serum and vaginal IgG and IgA responses. Our results show that HIV-gp140-carrying wax nanoparticles can induce strong cellular/humoral immune responses without inflammation and may be of potential use as effective mucosal adjuvants for HIV vaccine candidates.     

Recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant enhances induction of diphtheria and tetanus antitoxin antibodies in mice by intranasal administration with diphtheria-pertussis-tetanus (DPT) combination vaccine

Recombinant cholera toxin B subunit (rCTB) which is produced by Bacillus brevis carrying pNU212-CTB acts as a mucosal adjuvant capable of enhancing host immune responses specific to unrelated, mucosally co-administered vaccine antigens. When mice were administered intranasally with diphtheria-pertussis-tetanus (DPT) combination vaccine consisting of diphtheria toxoid (DTd), tetanus toxoid (TTd), pertussis toxoid (PTd), and formalin-treated filamentous hemagglutinin (fFHA), the presence of rCTB elevated constantly high values of DTd- and TTd-specific serum ELISA IgG antibody titres, and protective levels of diphtheria and tetanus toxin-neutralizing antibodies but the absence of rCTB did not. Moreover, the addition of rCTB protected all mice against tetanic symptoms and deaths. DPT combination vaccine raised high levels of serum anti-PT IgG antibody titres regardless of rCTB and protected mice from Bordetella pertussis challenge. These results suggest that co-administration of rCTB as an adjuvant is necessary for induction of diphtheria and tetanus antitoxin antibodies on the occasion of intranasal administration of DPT combination vaccine.     

A novel intranasal Protollin-based measles vaccine induces mucosal and systemic neutralizing antibody responses and cell-mediated immunity in mice

Protollin-MV is a vaccine produced by mixing split measles virus (MV) antigen with the novel adjuvant Protollin (Neisseria meningitidis outer membrane proteins non-covalently complexed with Shigella flexneri 2a lipopolysaccharide). Intranasal immunization of mice with two or three doses of Protollin-MV induces both serum IgG and mucosal IgA with strong neutralizing activity. There is a dose-dependent shift towards lower IgG1:IgG2a ratios and MV-specific IFNgamma production in splenocytes. Intranasal Protollin-MV can therefore induce systemic and mucosal neutralizing antibody responses as well as elicit a balanced TH1/TH2-type response.     

Alanyl-glutamine enriched total parenteral nutrition improves local, systemic, and remote organ responses to intraperitoneal bacterial challenge.

BACKGROUND: Standard total parenteral nutrition (STD-TPN) may diminish host defense against infection. Glutamine (Gln) is suggested to enhance host immunity. This study investigated the effects of antecedent alanyl-glutamine enriched TPN (Ala-Gln-TPN) on host responses to intraperitoneal bacterial challenge compared with STD-TPN. METHODS: Rats were divided into STD-TPN and Ala-Gln-TPN groups. They received isocaloric and isonitrogenous nutrition for 7 days and were challenged intraperitoneally with E. coli. Rats were killed before (0 hour) challenge and at 2 and 6 hours after challenge. Bacterial numbers in peritoneal lavage fluid (PLF), liver, spleen, and blood were determined. Tumor necrosis factor-alpha (TNF), interleukin (IL)-8, and interferon-gamma (IFN) in plasma and PLF were measured. Hepatic TNF, splenic TNF, and splenic IFN levels were determined. RESULTS: The numbers of E. coli in systemic blood at 2 hours after intraperitoneal bacterial challenge were significantly lower in the Ala-Gln-TPN than in STD-TPN group. E. coli numbers in blood significantly correlated with those in the liver. The Ala-Gln-TPN also resulted in significantly higher PLF and hepatic TNF levels, higher splenic IFN levels, and lower plasma IL-8 levels at 6 hours after challenge compared with the STD-TPN. CONCLUSIONS: Antecedent Ala-Gln enriched TPN enhance local, systemic, and remote organ immune responses to intraperitoneal bacterial challenge. Ala-Gln-TPN may enhance host defense and be more beneficial than standard TPN in sepsis.     

A novel dry powder influenza vaccine and intranasal delivery technology: induction of systemic and mucosal immune responses in rats

Intranasal (i.n.) vaccination represents an attractive non-invasive alternative to needle-based injection and provides superior protection at mucosal surfaces. However, new formulations are needed to improve efficacy and reduce the refrigerated storage and distribution requirements associated with standard liquid vaccines. Here, we describe a powder formulation of whole inactivated influenza virus and a novel i.n. delivery platform. The powder-formulated vaccine elicited a significant serum antibody response in rats that was at least as strong as that provided by the liquid vaccine administered i.n. or via intramuscular (i.m.) injection. Significant nasal IgA responses were also observed solely after i.n. delivery. This study demonstrates for the first time the generation of potent nasal mucosal and systemic immune responses using an i.n. delivered influenza vaccine powder and suggests an alternative approach to vaccination against influenza and other infectious diseases.     

Role of IgA in the defense against respiratory infections IgA deficient mice exhibited increased susceptibility to intranasal infection with Mycobacterium bovis BCG

IgA is the predominant Ig isotype in mucosal tissue and is believed to be involved in defense against viral and bacterial infections at these sites. Here, we examined the role of IgA in the protection against intranasal (i.n.) infection with Mycobacterium bovis Bacillus Calmette-Guerin (BCG). IgA deficient (IgA-/-) mice and wild type non-targeted littermate (IgA+/+) mice were immunized by i.n. route with the mycobacterium surface antigen PstS-1 formulated with cholera toxin (CT). Our data showed that IgA-/- mice were more susceptible to BCG infection compared to IgA+/+ mice, as revealed by the higher bacterial loads in the lungs and bronchoalveolar lavage (BAL). Analysis of the Ig levels and the antibody responses to PstS-1 showed that IgA-/- mice had no detectable IgA either in the saliva or in the BAL. However, these mice displayed higher levels of total and specific IgM than IgA+/+ mice in both mucosal fluids. More importantly, analysis of the cytokine responses revealed a reduction in the IFN-gamma and TNF-alpha production in the lungs of IgA-/- compared to IgA+/+ mice. Altogether, our results suggest that IgA may play a role in protection against mycobacterial infections in the respiratory tract by blocking the pathogen entrance and/or by modulating the pro-inflammatory responses.     

Frequent nasal administrations of recombinant cholera toxin B subunit (rCTB)-containing tetanus and diphtheria toxoid vaccines induced antigen-specific serum and mucosal immune responses in the presence of anti-rCTB antibodies

Vaccination via a mucosal route is a very attractive means for immunization, because both local and systemic immune responses are inducible and vaccines can be administered easily and safely from infants to elderly persons. For developing widely applicable mucosal vaccines using recombinant cholera toxin B subunit (rCTB) as a safe adjuvant, we examined whether frequent nasal administrations of rCTB-containing same and different vaccines could induce antigen-specific immune responses without induction of systemic tolerance and suppression by pre-existing anti-rCTB immunity. Ten repetitive nasal administrations to mice of tetanus toxoid (TT) + rCTB or diphtheria toxoid (DT) + rCTB raised and maintained high levels of antigen- and rCTB-specific serum IgG including high levels of tetanus/diphtheria antitoxin titres and raised nasal, salivary, lung, vaginal and fecal secreted IgA, suggesting that the regimen did not induce systemic tolerance to TT/DT and rCTB. Mice successively received repetitive five doses of TT as the first antigen and subsequent five doses of DT as the second antigen, and vice versa, raised serum IgG to the second antigen at various levels including low but sufficient protective levels of antitoxin titres and induced mucosal IgA in the lungs, the vaginas and feces, but hardly in the nasal secretions and salivas. After an interval of 22 weeks between the dosage of the first and second antigens, mice induced serum IgG to the second antigen at high levels and mucosal IgA in all sites. In conclusion, anti-TT and -DT serum and mucosal antibody responses induced by repeated intranasal immunization using rCTB adjuvant lasted for a long period, and for improving the effectivity of vaccination, different rCTB-containing vaccines should be administered at appropriate intervals.     

Fragilysin, the enterotoxin from Bacteroides fragilis, enhances the serum antibody response to antigen co-administered by the intranasal route

Fragilysin, an extracellular zinc metalloprotease produced by enterotoxigenic strains of the anaerobic bacterium Bacteroides fragilis, disrupts the paracellular barrier by cleavage of the intercellular proteins between epithelial cells resulting in fluid secretion. Intranasal immunization of mice with fragilysin and co-administered ovalbumin (Ova) resulted in an Ova-specific serum IgG response that was over 18000-fold higher than Ova alone, as well as detectable levels of serum IgA. Serum IgG titers were comparable with those seen when whole cholera toxin was used as the adjuvant, although the responses obtained with fragilysin showed more variability between mice. Metalloproteases to which fragilysin is structurally related were ineffective as mucosal adjuvants. Our results and similar studies with enterotoxins that affect the paracellular barrier suggest that alteration of mucosal permeability may play an important role in the mechanisms of adjuvanticity.     

The HIV-1 matrix protein p17 can be efficiently delivered by intranasal route in mice using the TLR 2/6 agonist MALP-2 as mucosal adjuvant

The HIV-1 matrix protein p17 is a structural protein essential in the life cycle of HIV, by acting as a virokine/immunomodulator that supports viral replication and spreading. The presence of p17-specific antibodies and CTL responses correlates with slower progression to AIDS. Intranasal vaccination with p17 and the TLR2/6 agonist MALP-2 stimulates strong humoral and cellular immune responses at systemic and mucosal levels. The antibodies blocked p17 binding to its receptor, which is a critical step for the exertion of its virokine activity. Our results suggest that p17 and MALP-2 are attractive candidates for incorporation in mucosal vaccines against HIV/AIDS.     

Enhanced humoral and mucosal immune responses after intranasal immunization with chimeric multiple antigen peptide of LcrV antigen epitopes of Yersinia pestis coupled to palmitate in mice

Yersinia pestis is the causative agent of the most deadly disease plague. F1 and V antigens are the major vaccine candidates. Six protective epitopes of V antigen of varying length (15-25aa) were assembled on a lysine backbone as multiple antigen peptide (MAP) using standard Fmoc chemistry. Palmitate was coupled at amino terminus end. Amino acid analysis, SDS-PAGE, immunoblot and immunoreactivity proved the authenticity of MAP. MAP was immunized intranasally encapsulated in PLGA (polylactide-co-glycolide) microspheres and with/without/adjuvants murabutide and CpG ODN 1826 (CpG), in three strains of mice. Humoral and mucosal immune responses were studied till day 120 and memory response was checked after immunization with native V antigen on day 120. Epitope specific serum and mucosal washes IgG, IgA, IgG subclasses and specific activity were measured by indirect ELISA and sandwich ELISA, respectively. IgG and IgA peak antibody titers of all the MAP construct formulations in sera were ranging from 71,944 to 360,578 and 4493 to 28,644, respectively. MAP with CpG showed significantly high (p < 0.0001) antibody titers ranging from 101,690 to 360,578 for IgG and 28,644 for IgA. Mucosal peak IgG and IgA titers were ranging from 1425 to 8072 and 1425 to 7183, respectively in intestinal washes and 799-4528 and 566-4027, respectively in lung washes. MAP with CpG showed significantly high (p < 0.001) SIgA titers of 8000 in lung and 16,000 in intestinal washes. IgG isotyping revealed IgG2a/IgG1 ratio > 1 with CpG. Serum and mucosal antipeptide IgG and IgA specific activities correlated well with antibody titers. All the constituent peptides contributed towards immune response. Structural analysis of MAP revealed little or no interaction between the peptides. Present study showed MAP to be highly immunogenic with high and long lasting antibody titers in serum and mucosal washes with good recall response with/without CpG as an adjuvant which can be used for vaccine development for plague.     

Isotype-specific antibody responses to foot-and-mouth disease virus in sera and secretions of "carrier' and "non-carrier' cattle.

Isotype-specific antibody responses to foot-and-mouth disease virus (FMDV) were measured in the sera and upper respiratory tract secretions of vaccinated and susceptible cattle challenged with FMDV by direct contact or by intranasal inoculation. A comparison was made between cattle that eliminated FMDV and those that developed and maintained a persistent infection. Serological and mucosal antibody responses were detected in all animals after challenge. IgA and IgM were detected before the development of IgG1 and IgG2 responses. IgM was not detected in vaccinated cattle. Challenge with FMDV elicited a prolonged biphasic secretory antibody response in FMDV "carrier'' animals only. The response was detected as FMDV-specific IgA in both mucosal secretions and serum samples, which gained statistical significance (P < 0.05) by 5 weeks after challenge. This observation could represent the basis of a test to differentiate vaccinated and/or recovered convalescent cattle from FMDV "carriers''.     

Effect of pre-existing immunity for systemic and mucosal immune responses to intranasal immunization with group B Streptococcus type III capsular polysaccharide-cholera toxin B subunit conjugate

The effects of priming with a group B Streptococcus type III capsular polysaccharide (GBS CPS III)-recombinant cholera toxin B subunit (rCTB) conjugate, purified GBS CPS III or rCTB alone on the systemic and mucosal immune responses to CPS III after intranasal (i.n.) immunization were investigated in mice. Priming with purified GBS CPS III followed by boosting with GBS CPS III-rCTB conjugate or priming with the conjugate followed by boosting with free CPS induced comparable levels of specific IgG and IgA in both serum and in lungs and vagina. However, i.n. immunization comprising both priming and boosting with conjugate was superior to priming with CPS and boosting with conjugate or the reverse, especially with regard to inducing mucosal IgA anti-CPS responses. All the immunization schemes, except priming and boosting with free CPS, induced high and similar levels of IgG1 in serum. In contrast, mice primed with free CPS III and then boosted with CPS III-rCTB conjugate by the i.n. route failed to produce significant levels of IgG2a, IgG2b and IgG3 in serum, at difference from mice primed with the conjugate and boosted with either conjugate or free CPS. Pre-immunization with rCTB either i.n. or i.p. did not suppress specific serum IgG responses induced by GBS CPS III-rCTB conjugate intranasally, but did inhibit serum and especially mucosal IgA responses. Our findings suggest that priming with CPS affects the distribution of IgG subclasses to GBS CPS and that pre-existing anti-carrier rCTB immunity can have an inhibitory effect on mucosal immune responses elicited by the conjugate vaccine given by the i.n. route.     

BAFF,stimulatory DNA and IL-15 stimulates IgA memory B cells and provides a novel approach for analysis of memory responses to mucosal vaccines

Assessment of immune responses induced by mucosal vaccines is to a large extent based on measurement of IgA levels in mucosal secretions and detection of short-lived effector IgA-secreting cells circulating in peripheral blood. Since these immunological parameters poorly reflect long-term IgA-mediated responses, we sought to investigate novel approaches that would enable detection of vaccine specific IgA memory B cells. We demonstrate that stimulation of human peripheral blood mononuclear cells in vitro with immunostimulatory DNA in combination with B cell-activating factor (BAFF) and IL-15 promotes differentiation of IgA memory B cells to IgA-secreting cells. By using the inactivated oral cholera vaccine Dukoral^® we demonstrate that vaccine specific IgA memory B cells are induced by oral immunization and are circulating for at least 9 months after vaccination. We also show that stimulated IgA memory B cells do not secrete IgA unless they reencounter the specific antigen.