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Cocaine is one of the most potent and addictive psychostimulants known and there are no available pharmacotherapies to treat cocaine addiction. Here we describe a novel cocaine vaccine employing the mucosal adjuvant and mast cell-activating oligopeptide, mastoparan-7 (M7), to achieve optimal IgA antibody responses in mucosal secretions and effective induction of humoral immunity using a short immunization protocol. This formulation, using a hapten-carrier system to deliver cocaine as antigen, also reduced cocaine penetration of the blood brain barrier and protected mice from its psychoactive effects by reducing cocaine-induced locomotion. Surprisingly, the magnitude of cocaine-specific antibody titers induced by each adjuvant was not the major determinant of functional protection from cocaine challenge. A side-by-side comparison of the two haptens, cocaine and its analog GNC demonstrated that cocaine haptenation resulted in superior functional protection when used in combination with the novel mucosal adjuvant, M7. These results provide a new potential strategy for combatting cocaine addiction through mucosal vaccination.

SARS-CoV-2 entry into cells requires specific host proteases; however, no successful in vivo applications of host protease inhibitors have yet been reported for treatment of SARS-CoV-2 pathogenesis. Here we describe a chemically engineered nanosystem encapsulating CRISPR–Cas13d, developed to specifically target lung protease cathepsin L (Ctsl) messenger RNA to block SARS-CoV-2 infection in mice. We show that this nanosystem decreases lung Ctsl expression in normal mice efficiently, specifically and safely. We further show that this approach extends survival of mice lethally infected with SARS-CoV-2, correlating with decreased lung virus burden, reduced expression of proinflammatory cytokines/chemokines and diminished severity of pulmonary interstitial inflammation. Postinfection treatment by this nanosystem dramatically lowers the lung virus burden and alleviates virus-induced pathological changes. Our results indicate that targeting lung protease mRNA by Cas13d nanosystem represents a unique strategy for controlling SARS-CoV-2 infection and demonstrate that CRISPR can be used as a potential treatment for SARS-CoV-2 infection.

SUMMARYThe COVID-19 pandemic persists as a global health crisis for which curative treatment has been elusive. Development of effective and safe anti-SARS-CoV-2 therapies remains an urgent need. SARS-CoV-2 entry into cells requires specific host proteases including TMPRSS2 and Cathepsin L (Ctsl)1–3, but there has been no reported success in inhibiting host proteases for treatment of SARS-CoV-2 pathogenesis in vivo. Here we have developed a lung Ctsl mRNA-targeted, CRISPR/Cas13d-based nanoparticle therapy to curb fatal SARS-CoV-2 infection in a mouse model. We show that this nanotherapy can decrease lung Ctsl expression in normal mice efficiently, specifically, and safely. Importantly, this lung-selective Ctsl-targeted nanotherapy significantly extended the survival of lethally SARS-CoV-2 infected mice by decreasing lung virus burden, reducing expression of pro-inflammatory cytokines/chemokines, and diminishing the severity of pulmonary interstitial inflammation. Additional in vitro analyses demonstrated that Cas13d-mediated Ctsl knockdown inhibited infection mediated by the spike protein of SARS-CoV-1, SARS-CoV-2, and more importantly, the authentic SARS-CoV-2 B.1.617.2 Delta variant, regardless of TMPRSS2 expression status. Our results demonstrate the efficacy and safety of a lung-selective, Ctsl-targeted nanotherapy against infection by SARS-CoV-2 and likely other emerging coronaviruses, forming a basis for investigation of this approach in clinical trials.

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of this research model are the ease of breeding and sample collection, relatively short gestation period, and large litter sizes. Commonly assessed routes of immunization include subcutaneous, intramuscular, intranasal, and intradermal. Nonterminal sample collection for the chronological detection of the immunologic responses to these immunizations include the collection of blood, from both dams and kits, and milk from the lactating does. In this article, we will demonstrate techniques our lab has utilized in studies of maternal immunization in New Zealand White rabbits (Oryctolagus cuniculus), including intranasal immunization and milk collection.

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of this research model are the ease of breeding and sample collection, relatively short gestation period, and large litter sizes. Commonly assessed routes of immunization include subcutaneous, intramuscular, intranasal, and intradermal. Nonterminal sample collection for the chronological detection of the immunologic responses to these immunizations include the collection of blood, from both dams and kits, and milk from the lactating does. In this article, we will demonstrate techniques our lab has utilized in studies of maternal immunization in New Zealand White rabbits (Oryctolagus cuniculus), including intranasal immunization and milk collection.

The development of mucosally administered vaccines remains a goal of many researchers who desire to develop a needle-free method of immunization that can induce antigen-specific immune responses in both systemic and mucosal tissues. The coadministration of adjuvants is often required to maximize the induction of protective immunity following mucosal vaccination, especially when subunit immunogens are used. A major class of mucosal vaccine adjuvants and modulators includes substances that can activate the innate immune system. Innate immune system activators that exhibit mucosal vaccine adjuvant and modulator activity will be discussed, including proinflammatory cytokines, nanoemulsions, mast cell activators, and toll-like receptor ligands.

Mast cells (MCs) are gaining recognition as key initiators and coordinators of host inflammatory and immune responses to various microbial pathogens. Their presence in mucosal tissues and skin make them one of the first immune cells to make contact with incoming pathogens. MCs can be readily activated at infection sites and release their collection of prestored and de novo synthesized mediators to initiate immune responses. Additionally, since MCs are in close proximity to blood vessels, they help to augment immune cell mobilization from the circulation to the infection site. However, not all MC reactions are beneficial, and if MCs become overactivated, as is observed with chronic or systemic infections, pathogenic sequelae ensue. In view of the powerful immunomodulatory role of MCs, there is growing interest in targeting these cells to either boost immunity or diminish harmful inflammatory outcomes of severe or chronic infection.

Mast cells are an important cell type of the innate immune system that when activated, play a crucial role in generating protective innate host responses after bacterial and viral infection. Additionally, activated mast cells influence lymph node composition to regulate the induction of adaptive immune responses. The recognition that mast cells play a beneficial role in host responses to microbial infection and induction of adaptive immunity has provided the rationale to evaluate mast cell activators for use as antimicrobials or vaccine adjuvants. This review summarizes the role of mast cell activators in antimicrobial responses while also discussing the use of different classes of mast cell activators as potent vaccine adjuvants that enhance the induction of protective immune responses.

Although animal research requires adherence to various regulations and standards, the manner in which compliance is maintained and the degree of additional constraints varies between institutions. Regulatory burden, particularly if institutionally imposed, has become a concern for institutions as increased regulatory expectations result in decreased resources available for research efforts. Faculty, research staff, and support staff engaged in animal research were surveyed to determine what institutional animal care and use committee (IACUC) processes were considered burdensome, the perceived value of some suggested modifications, and satisfaction with the IACUC administrative office and the animal resource unit. Although the results revealed overwhelming satisfaction with the IACUC administrative office and the animal resource unit, several IACUC processes were deemed burdensome, and therefore there would be value in modifying IACUC processes. When comparing the value of modifying IACUC processes, different groups within the animal care and use program (ACUP) tended to have different responses on many of the topics. This survey identified several perceived burdensome IACUC processes that would likely benefit individuals if modified. In today's environment of shrinking budgets for biomedical research, minimizing regulatory burden-particularly unnecessary, self-imposed burden-in the ACUP is particularly important to ensure that costs, time, and effort are appropriate to achieve animal welfare and quality of research endeavors.-Norton, J. N., Reynolds, R. P., Chan, C., Valdivia, R. H., Staats, H. F. Assessing the satisfaction and burden within an academic animal care and use program.

An effective adjuvant for the induction of humoural and cellular immunity is achieved by a Pickering emulsion formulation that allows pliability and mobility of loaded antigens.

Nonneutralizing antibody functions have been associated with reduced infection risk, or control of virus replication, for HIV-1 and related viruses. It is therefore critical to evaluate development of these responses throughout all stages of preclinical testing. Rabbits are conventionally used to evaluate the ability of vaccine candidates to safely elicit antibodies that bind and neutralize HIV-1. However, it remained unexplored how effectively rabbits model the development of nonneutralizing antibody responses in primates. We administered identical HIV-1 vaccine regimens to rabbits and rhesus macaques and performed detailed comparisons of vaccine-induced antibody responses. We demonstrated that nonneutralizing HIV-specific antibody responses can be studied in the rabbit model and have identified aspects of these responses that are common, and those that are unique, to rabbits and rhesus macaques. Our findings will help determine how to best utilize preclinical rabbit and rhesus macaque models to accelerate HIV vaccine candidate testing in human trials., Studies in animal models are essential prerequisites for clinical trials of candidate HIV vaccines. Small animals, such as rabbits, are used to evaluate promising strategies prior to further immunogenicity and efficacy testing in nonhuman primates. Our goal was to determine how HIV-specific vaccine-elicited antibody responses, epitope specificity, and Fc-mediated functions in the rabbit model can predict those in the rhesus macaque (RM) model. Detailed comparisons of the HIV-1-specific IgG response were performed on serum from rabbits and RM given identical modified vaccinia virus Ankara-prime/gp120-boost immunization regimens. We found that vaccine-induced neutralizing antibody, gp120-binding antibody levels and immunodominant specificities, antibody-dependent cellular phagocytosis of HIV-1 virions, and antibody-dependent cellular cytotoxicity (ADCC) responses against gp120-coated target cells were similar in rabbits and RM. However, we also identified characteristics of humoral immunity that differed across species. ADCC against HIV-infected target cells was elicited in rabbits but not in RM, and we observed differences among subdominantly targeted epitopes. Human Fc receptor binding assays and analysis of antibody-cell interactions indicated that rabbit vaccine-induced antibodies effectively recruited and activated human natural killer cells, while vaccine-elicited RM antibodies were unable to activate either human or RM NK cells. Thus, our data demonstrate that both Fc-independent and Fc-dependent functions of rabbit antibodies can be measured with commonly used in vitro assays; however, the ability of immunogenicity studies performed in rabbits to predict responses in RM will vary depending on the particular immune parameter of interest. IMPORTANCE Nonneutralizing antibody functions have been associated with reduced infection risk, or control of virus replication, for HIV-1 and related viruses. It is therefore critical to evaluate development of these responses throughout all stages of preclinical testing. Rabbits are conventionally used to evaluate the ability of vaccine candidates to safely elicit antibodies that bind and neutralize HIV-1. However, it remained unexplored how effectively rabbits model the development of nonneutralizing antibody responses in primates. We administered identical HIV-1 vaccine regimens to rabbits and rhesus macaques and performed detailed comparisons of vaccine-induced antibody responses. We demonstrated that nonneutralizing HIV-specific antibody responses can be studied in the rabbit model and have identified aspects of these responses that are common, and those that are unique, to rabbits and rhesus macaques. Our findings will help determine how to best utilize preclinical rabbit and rhesus macaque models to accelerate HIV vaccine candidate testing in human trials.

Mast cells (MCs) are known to regulate innate and adaptive immunity. MC activators have recently been described as safe and effective vaccine adjuvants. However, many currently known MC activators are inadequate for in vivo applications, and research on identifying novel MC activators is limited. In this study, we identified novel MC activators by employing high-throughput screening (HTS) assays using approximately 55,000 small molecules. Data sets obtained by the primary HTS assays were statistically evaluated using quality control rules and the B-score calculation, and compounds with B-scores of > 3.0 were chosen as mast cell activators (hits). These hits were re-evaluated with secondary and tertiary HTS assays, followed by further statistical analysis. From these hits, we selected 15 compounds that caused degranulation in murine and human MCs with potential for flexible chemical modification for further study. Among these 15 compounds, ST101036, ST029248 and ST026567 exhibited higher degranulation potency than other hit compounds in both human and mouse MCs. Additionally, the 15 compounds identified promote de novo synthesis of cytokines and induce the release of eicosanoids from human and mouse MCs. High-throughput screening enabled us to identify small molecule MC activators with unique properties that may be useful as vaccine adjuvants.

The benefits of mucosal vaccines over injected vaccines are difficult to ascertain since mucosally administered vaccines often induce serum antibody responses of lower magnitude than those induced by injected vaccines. This study aimed to determine if mucosal vaccination using a modified vaccinia Ankara expressing HIV-1 gp120 (MVA-g120) prime and HIV-1 gp120 protein boost could be optimized to induce serum antibody responses similar to those induced by an intramuscularly (IM) administered MVA prime/gp120 boost to allow comparison of an IM immunization regimen to a mucosal vaccination regimen for their ability to protect against a low dose rectal SHIV challenge while inducing similar serum anti-HIV-1 antibody responses. A 3-fold higher antigen dose was required for intranasal (IN) immunization with gp120 to induce serum anti-gp120 IgG responses not significantly different than those induced by IM immunization. Gp120 fused to the Adenovirus type 2 fiber binding domain (gp120-Ad2F), a mucosal targeting ligand, exhibited enhanced IN immunogenicity when compared to gp120 alone. MVA-gp120 was more immunogenic after IN delivery than gastric or rectal delivery, although serum antibodies induced by IN immunization were lower than those induced by intramuscular immunization. Using these optimized vaccines, an IN MVA-gp120 prime, combined IM (gp120) and IN (gp120-Ad2F) boost regimen (IN/IM+IN) induced serum anti-gp120 antibody titers similar to those induced by the intramuscular prime/boost regimen (IM/IM) in rabbits and non-human primates. Despite the induction of similar systemic anti-HIV-1 antibody responses, neither the IM/IM nor the IN/IM+IN regimen induced elevated anti-HIV-1 mucosal IgA responses nor protected against a repeated low-dose rectal SHIV challenge. These results demonstrate that immunization regimens utilizing the IN route are able to induce serum antigen-specific antibody responses similar to those induced by systemic immunizationIMPORTANCEMucosal vaccination is proposed as a method of immunization able to induce protection against mucosal pathogens that is superior to protection provided by parenteral immunization. However, mucosal vaccination often induces serum antigen-specific immune responses of lower magnitude than those induced by parenteral immunization, making the comparison of mucosal and parenteral immunization difficult. We identified vaccine parameters that allowed an immunization regimen consisting of an IN prime followed with boosters administered by both IN and IM routes to induce serum antibody responses similar to those induced by IM prime/boost vaccination. Additional studies are needed to determine the potential benefit of mucosal immunization for HIV-1 and other mucosally-transmitted pathogens.

Objective: Mast cell activators are a novel class of mucosal vaccine adjuvants. The polymeric compound, Compound 48/80 (C48/80), and cationic peptide, Mastoparan 7 (M7) are mast cell activators that provide adjuvant activity when administered by the nasal route. However, mast cell activating small molecules may be an adjuvant alternative that is easily synthesized with high purity compared to M7 or C48/80. The study objective is to identify small molecule mast cell activators that provide adjuvant activity in nasal West Nile virus vaccines. Methods: High-throughput screening assessed over 55,000 small molecules for mast cell degranulation activity. Fifteen hit compounds were down-selected to three by in vitro evaluation of immune activation activities, including cytokine production and cytotoxicity, and selection for functional diversity. These three molecules, ST101036, ST048871, and R529877, were then examined for their ability to evoke protection against West Nile virus infections in BALB/c mice when administered nasally with West Nile virus envelope domain III (EDIII) protein. Results: All three adjuvants evoked high levels of EDIII-specific antibody and conferred similar levels of protection, ST101036 (67%), ST048871 (73%) and R529877 (75%), which was also comparable to the protection induced by M7 (67%) but markedly higher than the levels seen with mice immunized with EDIII alone (no adjuvant 33%). Conclusion: Novel small molecule mast cell activators are as efficacious as previously described mast cell activating peptides and serve as potential replacements in in vivo studies.

The efficient encapsulation of therapeutic proteins into delivery vehicles, particularly without loss of function, remains a significant research hurdle. Typical liposomal formulations achieve drug loadings on the order of 3–5% and encapsulation efficiencies around 50%. We demonstrate the encapsulation of model proteins with isoelectric points above and below pH 7 into nanocarriers (NCs) with protein loadings as high as 46% and encapsulation efficiencies above 95%. This is done by combining the continuous nanofabrication process Flash NanoPrecipitation (FNP) with the technique of hydrophobic ion pairing by forming and encapsulating an ionic complex within a nanocarrier stabilized by a block copolymer surface layer. We complex and encapsulate lysozyme with two anionic hydrophobic counterions, sodium oleate and sodium dodecyl sulfate, using either a pre-formed complex or in situ pairing. The strategy successfully forms NCs ~150 nm in diameter and achieves encapsulation efficiencies over 95%. Protein release rate from the NCs in physiological conditions and the bioactivity of released lysozyme are measured, and both are found to vary with the complexing counterion and the protein/counterion ratio used during formulation. Protein release on the time scale of weeks is observed, and up to 100% bioactivity is measured from released lysozyme. 16 quaternary ammonium cationic counterions are tested to encapsulate ovalbumin in 32 formulations. Of these, 19 successfully form ~150 nm NCs with loadings up to 29% and encapsulation efficiencies up to 88%. We divide the formulations into four regimes and identify chemical factors responsible for the success or failure of a given counterion to formulate NCs with the desirable size, loading, and encapsulation efficiency. A successful ovalbumin NC formulation was then tested in vivo in a mouse nasal vaccine model and found to induce a higher titer of OVA-specific IgG than unencapsulated ovalbumin. Taken together, these findings suggest that Flash NanoPrecipitation with hydrophobic ion pairing is an attractive platform for encapsulating high molecular weight proteins into NCs. In particular, the ability to tune protein release rate by varying the counterion or protein/counterion ratio used during formulation is a useful feature.

Selective activation of local mast cells promotes healing of bacterial skin infections and protects against reinfection., Mast cells (MCs) are strategically distributed at barrier sites and prestore various immunocyte-recruiting cytokines, making them ideal targets for selective activation to treat peripheral infections. Here, we report that topical treatment with mastoparan, a peptide MC activator (MCA), enhances clearance of Staphylococcus aureus from infected mouse skins and accelerates healing of dermonecrotic lesions. Mastoparan functions by activating connective tissue MCs (CTMCs) via the MRGPRX2 (Mas-related G protein-coupled receptor member X2) receptor. Peripheral CTMC activation, in turn, enhances recruitment of bacteria-clearing neutrophils and wound-healing CD301b+ dendritic cells. Consistent with MCs playing a master coordinating role, MC activation also augmented migration of various antigen-presenting dendritic cells to draining lymph nodes, leading to stronger protection against a second infection challenge. MCAs therefore orchestrate both the innate and adaptive immune arms, which could potentially be applied to combat peripheral infections by a broad range of pathogens.

An injectable Vi-capsular polysaccharide vaccine against typhoid fever is available but vaccine-induced immunity tends to wane over time. The phenomenon of immunotolerance or hyporesponsiveness has earlier been described for polysaccharide vaccines such as pneumococcal capsular polysaccharide vaccine and some publications also suggest a possible immunotolerance after revaccination with Vi-capsular polysaccharide vaccines. In this study, post-immunisation antibody concentrations in adult travellers first vaccinated with a Salmonella typhi Vi-capsular polysaccharide vaccine (primary vaccination group) were compared with those having received one or more vaccinations previously (multiple vaccinations group). Vaccines administered were Typherix® (GlaxoSmithKline), Typhim Vi® (Sanofi Pasteur MSD) or Hepatyrix® (GlaxoSmithKline). Blood samples were obtained prior to vaccination (day 0) and on day 28 (−1/+14) after vaccination. Serum Vi-Antigen IgG concentrations were measured by ELISA. Of the 85 subjects included in the per protocol data set, 45 (53%) belonged to the multiple vaccinations group. In both groups, geometric mean antibody concentrations (GMCs) were significantly higher after vaccination than before vaccination. Pre-vaccination GMCs were lower in the primary vaccination group than in the multiple vaccinations group (3.40 μg/ml versus 6.13 μg/ml, P = 0.005), while there was no significant difference in the post vaccination GMCs between groups (11.34 μg/ml versus 14.58 μg/ml, P = 0.4). In the multiple vaccinations group, vaccination was performed 18 to 57 months after the last vaccination (median 38 months) and there was a negative correlation between time since last vaccination and antibody concentration on day 0. In conclusion, we were not able to demonstrate a relevant immunotolerance after multiple versus primary vaccination with S. typhi Vi-capsular polysaccharide vaccines.

INTRODUCTION An increasing number of individuals suffer from acute anaphylaxis, particularly food-associated reactions. The symptoms of anaphylaxis are triggered soon after allergens (such as peanut antigens, insect venom, or certain medications) enter the circulation of sensitized subjects who have elevated levels of antigen-specific immunoglobulin E (IgE) antibodies in their circulation. Mast cells (MCs) are the primary effectors of anaphylaxis and become activated when allergens make contact with IgE antibodies bound to the surfaces of MCs. Upon activation, MCs rapidly release prestored inflammatory mediators. When this occurs simultaneously throughout the body, anaphylaxis and shock result. As MCs are typically found in the perivascular abluminal surface of relatively impregnable blood vessels, how these cells perceive and interact with blood-borne allergens is unclear. RATIONALE Extravascular MCs were recently reported to be capable of extending cellular protrusions to directly sample vascular contents. Therefore, we reasoned that if MCs are directly sampling the blood, we should find that MCs are the first cells to acquire allergens upon systemic infusion of labeled allergen. We systemically infused mice with labeled dextran and examined which immune cells in the proximity of the vasculature had acquired the label within 30 min of antigen administration. RESULTS Surprisingly, MCs acquired only a small number of the dextran particles. Instead, most of the labeled antigen was acquired by a subset of dermal dendritic cells (DCs). These DCs, defined as CD301b + dermal cDC2, were found in close proximity to MCs in the perivascular abluminal surface lining dermal blood vessels. Furthermore, they appeared to directly sample the blood through the extrusion of dendrites. These DCs were critical to anaphylaxis, as IgE-sensitized mice deficient in CD301b + dermal cDC2 failed to undergo anaphylaxis. Live microscopy of the mouse vasculature revealed numerous perivascular DCs routinely sampling blood-borne allergens via dendrites, which penetrated the endothelial wall into the vascular lumen (left panel in the figure). Blood-borne antigens captured in this manner were relayed on the surfaces of 0.5- to 1.0-μm microvesicles (MVs) to neighboring MCs and DCs in the perivascular space. A panoply of receptors can be found on the surfaces of DCs, including the mannose receptor, which enable these cells to capture a broad range of foreign antigens in the blood. Knocking down mannose receptors markedly reduced the capacity of DCs to bind antigen and convey antigen to MCs. Furthermore, large numbers of MVs were constitutively shed by DCs regardless of whether contact with antigens was made. MVs were products of specific budding and pinching-off activities occurring on the plasma membranes of DCs. Additionally, MV shedding was abrogated when vacuolar protein sorting–associated protein 4 (VPS4), which mediates scission of MVs, was knocked down. DCs impaired in their ability to express VPS4 were found to be incapable of activating MCs or causing anaphylaxis. Noticeably, distribution of antigens by DCs on budding MVs occurred relatively quickly and was not preceded by internalization of antigen. Upon contact with allergens on the surfaces of MVs, IgE-bound MCs were found to degranulate vigorously, releasing their payload of inflammatory mediators (right panel in the figure) and triggering anaphylaxis. Thus, allergen-induced MC degranulation and anaphylaxis require the critical contribution of perivascular DCs in capturing and relaying circulating allergens. CONCLUSION We demonstrate how IgE-sensitized MCs are indirectly activated by blood-borne allergens. Additionally, our study reveals how perivascular DCs continuously sample blood and initiate and markedly enhance inflammatory and immune responses by rapidly discharging antigen-bearing MVs to surrounding immune cells.

The interdependent relationship between pharmacology and toxicology is fundamental to the concepts of efficacy and safety of both drugs and xenobiotics. The traditional concept of establishing efficacious and tolerated doses to define a 'therapeutic window' appears simplistic in the context of an exponentially increasing database on molecular mechanisms and cell biology that inform our understanding of homeostasis. Recent advances in nano medicine illustrate the convergence of efficacy and safety considerations that are central to establishing a clear pathway for regulatory review. The following overview considers biological responses to the administration of nanoparticles and the scale of balanced, within a range that might be considered 'normal', to unbalanced, abnormal responses associated with health and disease.

SummaryThe virulence of Salmonella is linked to its invasive capacity and suppression of adaptive immunity. This does not explain, however, the rapid dissemination of the pathogen after it breaches the gut. In our study, S. Typhimurium suppressed degranulation of local mast cells (MCs), resulting in limited neutrophil recruitment and restricting outflow of vascular contents into infection sites, thus facilitating bacterial spread. MC suppression was mediated by secreted effector protein (SptP), which shares structural homology with Yersinia YopH. SptP functioned by dephosphorylating the vesicle fusion protein N-ethylmalemide-sensitive factor and by blocking phosphorylation of Syk. Without SptP, orally challenged S. Typhimurium failed to suppress MC degranulation and exhibited limited colonization of the mesenteric lymph nodes. Administration of SptP to sites of E. coli infection markedly enhanced its virulence. Thus, SptP-mediated inactivation of local MCs is a powerful mechanism utilized by S. Typhimurium to impede early innate immunity.

Broad-spectrum antiviral drugs are urgently needed to treat individuals infected with new and re-emerging viruses, or with viruses that have developed resistance to antiviral therapies. Mammalian natural host defense peptides (mNHP) are short, usually cationic, peptides that have direct antimicrobial activity, and which in some instances activate cell-mediated antiviral immune responses. Although mNHP have potent activity in vitro, efficacy trials in vivo of exogenously provided mNHP have been largely disappointing, and no mNHP are currently licensed for human use. Mastoparan is an invertebrate host defense peptide that penetrates lipid bilayers, and we reasoned that a mastoparan analog might interact with the lipid component of virus membranes and thereby reduce infectivity of enveloped viruses. Our objective was to determine whether mastoparan-derived peptide MP7-NH2 could inactivate viruses of multiple types, and whether it could stimulate cell-mediated antiviral activity. We found that MP7-NH2 potently inactivated a range of enveloped viruses. Consistent with our proposed mechanism of action, MP7-NH2 was not efficacious against a non-enveloped virus. Pre-treatment of cells with MP7-NH2 did not reduce the amount of virus recovered after infection, which suggested that the primary mechanism of action in vitro was direct inactivation of virus by MP7-NH2. These results demonstrate for the first time that a mastoparan derivative has broad-spectrum antiviral activity in vitro and suggest that further investigation of the antiviral properties of mastoparan peptides in vivo is warranted.

Development of nasal immunization for human use is hindered by the lack of acceptable adjuvants. Although CT is an effective adjuvant, its toxicity will likely prevent its use in nasal vaccines. This study compared non-toxin adjuvants to CT for their ability to induce protective antibody responses with nasal immunization. C3H/HeN and C57BL/6 mice were immunized with rPA formulated with the following adjuvants: CT, IL-1α, LPS, CpG, Pam3CSK4, 3M-019, resiquimod/R848 or c48/80. Serum and nasal wash cytokine concentrations were monitored 6 h post-vaccination as biomarkers for acute activation of the innate immune system. Not all of the adjuvants induced significant changes in innate serum or nasal wash cytokines, but when changes were observed, the cytokine signatures were unique for each adjuvant. All adjuvants except Pam3CSK4 induced significantly increased anti-rPA serum IgG titers in both strains of mice, while only IL-1α, c48/80 and CpG enhanced mucosal anti-rPA IgA. Pam3CSK4 was the only adjuvant unable to enhance the induction of serum LeTx-neutralizing antibodies in C3H/HeN mice while c48/80 was the only adjuvant to induce increased serum LeTx-neutralizing antibodies in C57BL/6 mice. Only CT enhanced total serum IgE in C3H/HeN mice while IL-1α enhanced total serum IgE in C57BL/6 mice. The adjuvant influenced antigen-specific serum IgG subclass and T cell cytokine profiles, but these responses did not correlate with the induction of LeTx-neutralizing activity. Our results demonstrate the induction of diverse innate and adaptive immune responses by non-toxin nasal vaccine adjuvants that lead to protective humoral immunity comparable to CT and that these responses may be influenced by the host strain.

The development and use of vaccines and their ability to prevent infection/disease is a shining example of the benefit of biomedical research. Modern vaccines often utilize subunit immunogens that exhibit minimal immunogenicity and require the use of adjuvants to maximize the induction of protective immune responses. We recently described a novel class of vaccine adjuvants, mast cell (MC) activators, that exhibit safe and effective vaccine adjuvant activity when administered by intranasal or intradermal routes. A compound library containing 580 functionalized benzopyrans, a structural motif found in a diverse array of natural and designed bioactive compounds, was screened using a MC degranulation assay to identify novel MC activating compounds for future evaluation as novel vaccine adjuvants. This approach identified 12 novel MC degranulating compounds. Therefore, MC degranulation can be used to reliably detect novel compounds for evaluation as adjuvants for use in mucosal vaccine strategies.

Maternal vaccination to induce anti-HIV immune factors in breast milk is a potential intervention to prevent postnatal HIV-1 mother-to-child transmission (MTCT). We previously demonstrated that immunization of lactating rhesus monkeys with a modified vaccinia Ankara (MVA) prime/intramuscular (i.m.) protein boost regimen induced functional IgG responses in milk, while MVA prime/intranasal (i.n.) boost induced robust milk Env-specific IgA responses. Yet, recent studies have suggested that prevention of postnatal MTCT may require both Env-specific IgA and functional IgG responses in milk. Thus, to investigate whether both responses could be elicited by a combined systemic/mucosal immunization strategy, animals previously immunized with the MVA prime/i.n. boost regimen received an i.n./i.m. combined C.1086 gp120 boost. Remarkably, high-magnitude Env-specific IgA responses were observed in milk, surpassing those in plasma. Furthermore, 29% of vaccine-elicited Env-specific B cells isolated from breast milk were IgA isotype, in stark contrast to the overwhelming predominance of IgG isotype Env-specific B cells in breast milk of chronically HIV-infected women. A clonal relationship was identified between Env-specific blood and breast milk B cells, suggesting trafficking of that cell population between the two compartments. Furthermore, IgA and IgG monoclonal antibodies isolated from Env-specific breast milk B cells demonstrated diverse Env epitope specificities and multiple effector functions, including tier 1 neutralization, antibody-dependent cellular cytotoxicity (ADCC), infected cell binding, and inhibition of viral attachment to epithelial cells. Thus, maternal i.n./i.m. combined immunization is a novel strategy to enhance protective Env-specific IgA in milk, which is subsequently transferred to the infant via breastfeeding. IMPORTANCE Efforts to increase the availability of antiretroviral therapy to pregnant and breastfeeding women in resource-limited areas have proven remarkably successful at reducing HIV vertical transmission rates. However, more than 200,000 children are infected annually due to failures in therapy implementation, monitoring, and adherence, nearly half by postnatal HIV exposure via maternal breast milk. Intriguingly, in the absence of antiretroviral therapy, only 10% of breastfed infants born to HIV-infected mothers acquire the virus, suggesting the existence of naturally protective immune factors in milk. Enhancement of these protective immune factors through maternal vaccination will be a critical strategy to reduce the global pediatric AIDS epidemic. We have previously demonstrated that a high magnitude of HIV Env-specific IgA in milk correlates with reduced risk of infant HIV acquisition. In this study, we describe a novel HIV vaccine regimen that induces potent IgA responses in milk and therefore could potentially protect against breast milk HIV MTCT.

Capsule Summary Peanut-specific IgA in saliva correlates with DBPCFC outcomes following peanut SLIT, suggesting that peanut-specific salivary IgA may be a potential biomarker for SLIT used to treat peanut allergy.

IL-1 has been shown to have strong mucosal adjuvant activities, but little is known about its mechanism of action. We vaccinated IL-1R1 bone marrow (BM) chimeric mice to determine whether IL-1R1 expression on stromal cells or hematopoietic cells was sufficient for the maximal adjuvant activity of nasally delivered IL-1α as determined by the acute induction of cytokine responses and induction of Bacillus anthracis lethal factor (LF)-specific adaptive immunity. Cytokine and chemokine responses induced by vaccination with IL-1α were predominantly derived from the stromal cell compartment and included G-CSF, IL-6, IL-13, MCP-1, and keratinocyte chemoattractant. Nasal vaccination of Il1r1−/− (knock-out [KO]) mice given wild-type (WT) BM (WT→KO) and WT→WT mice with LF + IL-1α induced maximal adaptive immune responses, whereas vaccination of WT mice given Il1r1−/− BM (KO→WT) resulted in significantly decreased production of LF-specific serum IgG, IgG subclasses, lethal toxin-neutralizing Abs, and mucosal IgA compared with WT→KO and WT→WT mice (p < 0.05). IL-1α adjuvant activity was not dependent on mast cells. However, the ability of IL-1α to induce serum LF-specific IgG2c and lethal toxin-neutralizing Abs was significantly impaired in CD11c-Myd88−/− mice when compared with WT mice (p < 0.05). Our results suggest that CD11c+ cells must be directly activated by nasally administered IL-1α for maximal adjuvant activity and that, although stromal cells are required for maximal adjuvant-induced cytokine production, the adjuvant-induced stromal cell cytokine responses are not required for effective induction of adaptive immunity.

Due to its potential as an effective, needle-free route of immunization for use with subunit vaccines, nasal immunization continues to be evaluated as a route of immunization in both research and clinical studies. However, as with other vaccination routes, subunit vaccines often require the addition of adjuvants to induce potent immune responses. Unfortunately, many commonly used experimental vaccine adjuvants, such as cholera toxin andE. coliheat-labile toxin, are too toxic for use in humans. Because new adjuvants are needed, cytokines have been evaluated for their ability to provide effective adjuvant activity when delivered by the nasal route in both animal models and in limited human studies. It is the purpose of this paper to discuss the potential of cytokines as nasal vaccine adjuvants.

IL-1α and IL-1β were evaluated for their ability to provide adjuvant activity for the induction of serum antibody responses when nasally-administered with protein antigens in mice and rabbits. In mice, intranasal (i.n.) immunization with pneumococcal surface protein A (PspA) or tetanus toxoid (TT) combined with IL-1β induced protective immunity that was equivalent to that induced by parenteral immunization. Nasal immunization of awake (i.e., not anesthetized) rabbits with IL-1-adjuvanted vaccines induced highly variable serum antibody responses and was not as effective as parenteral immunization for the induction of antigen-specific serum IgG. However, i.n. immunization of deeply anesthetized rabbits with rPA + IL-1α consistently induced rPA-specific serum IgG ELISA titers that were not significantly different than those induced by intramuscular (IM) immunization with rPA + alum although lethal toxin neutralizing titers induced by nasal immunization were lower than those induced by IM immunization. Gamma scintigraphy demonstrated that the enhanced immunogenicity of nasal immunization in anesthetized rabbits correlated with an increased nasal retention of i.n. delivered non-permeable radio-labeled colloidal particles. Our results demonstrate that, in mice, IL-1 is an effective adjuvant for nasally-administered vaccines for the induction of protective systemic immunity and that in non-rodent species, effective induction of systemic immunity with nasally-administered vaccines may require formulations that ensure adequate retention of the vaccine within the nasal cavity.

Haemophilus ducreyi , the etiological agent of chancroid, has a strict requirement for heme, which it acquires from its only natural host, humans. Previously, we showed that a vaccine preparation containing the native hemoglobin receptor HgbA purified from H. ducreyi class I strain 35000HP (nHgbA I ) and administered with Freund's adjuvant provided complete protection against a homologous challenge. In the current study, we investigated whether nHgbA I dispensed with monophosphoryl lipid A (MPL), an adjuvant approved for use in humans, offered protection against a challenge with H. ducreyi strain 35000HP expressing either class I or class II HgbA (35000HP hgbA I and 35000HP hgbA II , respectively). Pigs immunized with the nHgbA I /MPL vaccine were protected against a challenge from homologous H. ducreyi strain 35000HP hgbA I but not heterologous strain 35000HP hgbA II , as evidenced by the isolation of only strain 35000HP hgbA II from nHgbA I -immunized pigs. Furthermore, histological analysis of the lesions showed striking differences between mock-immunized and nHgbA I -immunized animals challenged with strains 35000HP hgbA I but not those challenged with strain 35000HP hgbA II . Mock-immunized pigs were not protected from a challenge by either strain. The enzyme-linked immunosorbent assay (ELISA) activity of the nHgbA I /MPL antiserum was lower than the activity of antiserum from animals immunized with the nHgbA I /Freund's vaccine; however, anti-nHgbA I from both studies bound whole cells of 35000HP hgbA I better than 35000HP hgbA II and partially blocked hemoglobin binding to nHgbA I . In conclusion, despite eliciting lower antibody ELISA activity than the nHgbA I /Freund's, the nHgbA I /MPL vaccine provided protection against a challenge with homologous but not heterologous H. ducreyi , suggesting that a bivalent HgbA vaccine may be needed.

Vi polysaccharide from Salmonella enterica serotype Typhi is used as one of the available vaccines to prevent typhoid fever. Measurement of Vi-specific serum antibodies after vaccination with Vi polysaccharide by enzyme-linked immunosorbent assay (ELISA) may be complicated due to poor binding of the Vi polysaccharide to ELISA plates resulting in poor reproducibility of measured antibody responses. We chemically conjugated Vi polysaccharide to fluorescent beads and performed studies to determine if a bead-based immunoassay provided a reproducible method to measure vaccine-induced anti-Vi serum IgG antibodies. Compared to ELISA, the Vi bead immunoassay had a lower background and therefore a greater signal-to-noise ratio. The Vi bead immunoassay was used to evaluate serum anti-Vi IgG in 996 subjects from the city of Kolkata, India, before and after vaccination. Due to the location being one where Salmonella serotype Typhi is endemic, approximately 45% of the subjects had protective levels of anti-Vi serum IgG (i.e., 1 μg/ml anti-Vi IgG) before vaccination, and nearly 98% of the subjects had protective levels of anti-Vi serum IgG after vaccination. Our results demonstrate that a bead-based immunoassay provides an effective, reproducible method to measure serum anti-Vi IgG responses before and after vaccination with the Vi polysaccharide vaccine.

During infection, signals from the periphery are known to reach draining lymph nodes (DLNs), but how these molecules, such as inflammatory cytokines, traverse the significant distances involved without dilution or degradation remains unclear. We show that peripheral mast cells, upon activation, release stable submicrometer heparin-based particles containing tumor necrosis factor and other proteins. These complexes enter lymphatic vessels and rapidly traffic to the DLNs. This physiological drug delivery system facilitates communication between peripheral sites of inflammation and remote secondary lymphoid tissues.

SummaryMast cells (MCs) are best known for eliciting harmful reactions, mostly after primary immunity has been established. Here, we report that, during footpad infection with E. coli in MC-deficient mice, as compared to their MC-sufficient counterparts, the serum antibody response is significantly diminished and less protective following passive immunization in a urinary tract infection (UTI) model in wild-type mice. MCs were found to recruit large numbers of dendritic cells (DCs) into the infected tissue site, which eventually migrated into draining lymph nodes (DLNs) during a prolonged time course. This pattern of trafficking was facilitated by MC-generated TNF, which increased the expression of E-selectin on local blood vessels. Antibody blockade of E-selectin inhibited DC recruitment into the site of infection and DLNs and consequently impaired the primary humoral immune response. Thus, during infection, resident MCs contribute to the primary protective adaptive response through recruitment of DCs from the circulation into infected sites.

Alphavirus replicon particles induce strong antibody and CD8+ T cell responses to expressed antigens in numerous experimental systems. We have recently demonstrated that Venezuelan equine encephalitis virus replicon particles (VRP) possess adjuvant activity for systemic and mucosal antibody responses. In this report, we demonstrate that VRP induced an increased and balanced serum IgG subtype response to co-delivered antigen, with simultaneous induction of antigen-specific IgG1 and IgG2a antibodies, and increased both systemic and mucosal antigen-specific CD8+ T cell responses, as measured by an IFN-gamma ELISPOT assay. Additionally, VRP further increased antigen-specific T cell immunity in an additive fashion following co-delivery with the TLR ligand, CpG DNA. VRP infection led to recruitment of CD8+ T cells into the mucosal compartment, possibly utilizing the mucosal homing receptor, as this integrin was upregulated on CD8+ T cells in the draining lymph node of VRP-infected animals, where VRP-infected dendritic cells reside. This newly recognized ability of VRP to mediate increased T cell response towards co-delivered antigen provides the potential to both define the molecular basis of alphavirus-induced immunity, and improve alphavirus-based vaccines.

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.

Passive transfer of antibody may be useful for preexposure prophylaxis against biological agents used as weapons of terror, such asBacillus anthracis. Studies were performed to evaluate the ability of anthrax antiprotective antigen (anti-PA) and antilethal factor (anti-LF) neutralizing monoclonal antibodies (mAbs) to protect against an anthrax lethal toxin (LeTx) challenge in a mouse model and to identify correlates of immunity to LeTx challenge. Despite having similar affinities for their respective antigens, anti-PA (3F11) and anti-LF (9A11), passive transfer of up to 1.5 mg of anti-PA 3F11 mAb did not provide significant protection when transferred to mice 24 h before LeTx challenge, while passive transfer of as low as 0.375 mg of anti-LF 9A11 did provide significant protection. Serum collected 24 h after passive transfer had LeTx-neutralizing activity when tested using a standard LeTx neutralization assay, but neutralization titers measured using this assay did not correlate with protection against LeTx challenge. However, measurement of LeTx-neutralizing serum responses with an LeTx neutralization assay in vitro employing the addition of LeTx to J774A.1 cells 15 min before the addition of the serum did result in neutralization titers that correlated with protection against LeTx challenge. Our results demonstrate that only the LeTx neutralization titers measured utilizing the addition of LeTx to J774A.1 cells 15 min before the addition of sample correlated with protection in vivo. Thus, this LeTx neutralization assay may be a more biologically relevant neutralization assay to predict the in vivo protective capacity of LeTx-neutralizing antibodies.

Botulism has classically been considered to be a food- and water-borne disease. However, it was recently classified by the US National Institute of Allergy and Infectious Diseases (National Institute of Health) and the US Centers for Disease Control and Prevention as a Category A agent. Thus, the botulinum exotoxin, a neurotoxin, could be easily disseminated by bioterrorists through the air-borne route with a high morbidity and mortality rate. In this regard, a high priority should be given to the development of a safe and effective mucosal vaccine to protect against botulinum neurotoxins (BoNTs) since it is well known that the mucosal immune system is the first line of defense against major pathogens. Further, mucosal immunization has been shown to induce both mucosal and systemic immunity to pathogens. By contrast, the current injection-type vaccine only provides protective immunity in the systemic compartment. Clearly, the development of a safe and effective mucosal vaccine against this toxin should be a high priority. In this regard, it has been shown that both nasal and oral immunization approaches have been taken in order to protect from BoNT intoxication. In this article, we will discuss the importance of the development of a mucosal vaccine against botulinum and introduce current aspects of BoNT mucosal vaccines, which show that they effectively prevent mucosal BoNT intoxication.