Secretory IgA and course of COVID-19 in patients receiving a bacteria-based immunostimulant agent in addition to background therapy.

Mucosal immunity plays a major role not only in the prevention but probably also in the outcomes of COVID-19. An enhanced production of secretory immunoglobulin A (sIgA) might contribute to the activation of the immune response mechanisms. To assess the levels of sIgA produced by epithelial cells in the nasal and pharyngeal mucosa and those measured in salivary gland secretions and to study the course of COVID-19 following the combined scheme of intranasal and subcutaneous administration of a bacteria-based immunostimulant agent. This study included 69 patients, aged between 18 and 60, who had moderate COVID-19 infection. They were divided into two groups: Group 1 (control group) included 39 patients who received only background therapy, and Group 2 was made up of 30 patients who received background therapy in combination with the Immunovac VP4 vaccine, a bacteria-based immunostimulant agent, which was given for 11 days starting from the day of admission to hospital. The levels of sIgA were measured by ELISA in epithelial, nasal and pharyngeal swabs, and salivary gland secretions at baseline and on days 14 and 30. The combined scheme of intranasal and subcutaneous administration of the Immunovac VP4 vaccine in the complex therapy of patients with COVID-19 is accompanied by increased synthesis of sIgA in nasal and pharyngeal swabs, more intense decrease in the level of C-reactive protein (CRP) and reduction in the duration of fever and length of hospitalization compared to the control group. Prescribing a immunostimulant agent containing bacterial ligands in complex therapy for COVID-19 patients helps to enhance mucosal immunity and improves the course of the disease.

Preparation and characterization of curdlan-chitosan conjugate nanoparticles as mucosal adjuvants for intranasal influenza H1N1 subunit vaccine.

Intranasal vaccination offers crucial protection against influenza virus pandemics. However, antigens, especially subunit antigens, often fail to induce effective immune responses without the help of immune adjuvants. Our research has demonstrated that a polyelectrolyte complex, composed of curdlan sulfate/O-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (CS/O-HTCC), effectively triggers both mucosal and systemic immune responses when administrated intranasal. In this study, stable nanoparticles formed by curdlan-O-HTCC conjugate (CO NP) were prepared and characterized. Furthermore, the efficacy of CO NP was evaluated as a mucosal adjuvant in an intranasal influenza H1N1 subunit vaccine. The results revealed that CO NP exhibits uniform and spherical morphology, with a size of 190.53 ± 4.22 nm, and notably, it remains stable in PBS at 4 °C for up to 6 weeks. Biological evaluation demonstrated that CO NP stimulates the activation of antigen-presenting cells (APCs), including macrophages and dendritic cells (DCs), both in vitro and in vivo. Furthermore, intranasal administration of CO NP effectively elicits cellular and humoral immune responses, notably enhancing mucosal immunity. Thus, CO NP emerges as a promising mucosal adjuvant for influenza subunit vaccines.

Size effect of mesoporous silica nanoparticles on regulating the immune effect of oral influenza split vaccine.

Mucosal immunization is a powerful weapon against viral infection. In this paper, large pore mesoporous silica nanoparticles (LMSN) with different particle sizes were synthesized for loading influenza split vaccine (SV) to explore the effect of nanoparticle sizes on mucosal immunization and adjuvant efficacy. Interestingly, it was found that among the three particle sizes of nanoparticles, only LMSN-M with around 250 nm could significantly enhance the mucosal immune effect of SV, possessing adjuvant effect. The results indicated that particle size affected the adjuvant effect of LMSN. There was no apparent difference in vaccine loading capacity of LMSN with different particle sizes, but the release of SV depended on the pore length of LMSN. The adjuvant effect of LMSN-M was attributed to its higher cellular uptake performance, intestine absorption and transport efficiency, and the ability to stimulate the maturation of dendritic cells. Simultaneously, compared with LMSN-S and LMSN-L, the more retention of LMSN-M in mesenteric lymph nodes increased the chance of interaction between vaccine and immune system, resulting in the enhanced immunity. This is the first time to study the impact of particle size of LMSN adjuvant on improving mucosal immunity of oral influenza vaccine, and the present work provides a scientific reference for adjuvant design of oral vaccine.

Injectable Hydrogel Mucosal Vaccine Elicits Protective Immunity against Respiratory Viruses.

Intranasal vaccines, eliciting mucosal immune responses, can prevent early invasion, replication, and transmission of pathogens in the respiratory tract. However, the effective delivery of antigens through the nasal barrier and boosting of a robust systematic and mucosal immune remain challenges in intranasal vaccine development. Here, we describe an intranasally administered self-healing hydrogel vaccine with a reversible strain-dependent sol-gel transition by precisely modulating the self-assembly processes between the natural drug rhein and aluminum ions. The highly bioadhesive hydrogel vaccine enhances antigen stability and prolongs residence time in the nasal cavity and lungs by confining the antigen to the surface of the nasal mucosa, acting as a "mucosal mask". The hydrogel also stimulates superior immunoenhancing properties, including antigen internalization, cross-presentation, and dendritic cell maturation. Furthermore, the formulation recruits immunocytes to the nasal mucosa and nasal-associated lymphoid tissue (NALT) while enhancing antigen-specific humoral, cellular, and mucosal immune responses. Our findings present a promising strategy for preparing intranasal vaccines for infectious diseases or cancer.

Engineered Nanoparticles inside a Microparticle Oral System for Enhanced Mucosal and Systemic Immunity.

Antigen delivery through an oral route requires overcoming multiple challenges, including gastrointestinal enzymes, mucus, and epithelial tight junctions. Although each barrier has a crucial role in determining the final efficiency of the oral vaccination, transcytosis of antigens through follicle-associated epithelium (FAE) represents a major challenge. Most of the research is focused on delivering an antigen to the M-cell for FAE transcytosis because M-cells can easily transport the antigen from the luminal site. However, the fact is that the M-cell population is less than 1% of the total gastrointestinal cells, and most of the oral vaccines have failed to show any effect in clinical trials. To challenge the current dogma of M-cell targeting, in this study, we designed a novel tandem peptide with a FAE-targeting peptide at the front position and a cell-penetrating peptide at the back position. The tandem peptide was attached to a smart delivery system, which overcomes the enzymatic barrier and the mucosal barrier. The result showed that the engineered system could target the FAE (enterocytes and M-cells) and successfully penetrate the enterocytes to reach the dendritic cells located at the subepithelium dome. There was successful maturation and activation of dendritic cells in vitro confirmed by a significant increase in maturation markers such as CD40, CD86, presentation marker MHC I, and proinflammatory cytokines (TNF-α, IL-6, and IL-10). The in vivo results showed a high production of CD4+ T-lymphocytes (helper T-cell) and a significantly higher production of CD8+ T-lymphocytes (killer T-cell). Finally, the production of mucosal immunity (IgA) in the trachea, intestine, and fecal extracts and systemic immunity (IgG, IgG1, and IgG2a) was successfully confirmed. To the best of our knowledge, this is the first study that designed a novel tandem peptide to target the FAE, which includes M-cells and enterocytes rather than M-cell targeting and showed that a significant induction of both the mucosal and systemic immune response was achieved compared to M-cell targeting.

Metformin ameliorates chronic colitis in a mouse model by regulating interferon-γ-producing lamina propria CD4+ T cells through AMPK activation.

Metformin, a commonly prescribed drug for type 2 diabetes mellitus, has been shown to activate AMP-activated protein kinase (AMPK). Notably, AMPK activation has recently been observed to be associated with anti-inflammatory responses. Metformin is also reported to elicit anti-inflammatory responses in CD4+ T cells, resulting in improvement in experimental chronic inflammatory diseases, such as systemic lupus erythematosus. To investigate the effect of metformin on inflammatory bowel disease (IBD), we developed a T cell-transfer model of chronic colitis in which SCID mice were injected with CD4+ CD45RBhigh T cells to induce colitis. We examined the effects of metformin via in vitro and in vivo experiments on lamina propria (LP) CD4+ T cells. We observed that metformin suppresses the frequency of interferon (IFN) -γ-producing LP CD4+ T cells in vitro, which were regulated by AMPK activation, a process possibly induced by the inhibition of oxidative phosphorylation. Furthermore, we examined the effects of metformin on an in vivo IBD model. Metformin-treated mice showed AMPK activation in LP CD4+ T cells and ameliorated colitis. Our study demonstrates that metformin-induced AMPK activation in mucosal CD4+ T cells contributes to the improvement of IBD by suppressing IFN-γ production. Moreover, our results indicate that AMPK may be a target molecule for the regulation of mucosal immunity and inflammation. Thus, AMPK-activating drugs such as metformin may be potential therapeutic agents for the treatment of IBD.

Aerosol delivery, but not intramuscular injection, of adenovirus-vectored tuberculosis vaccine induces respiratory-mucosal immunity in humans.

BackgroundAdenovirus-vectored (Ad-vectored) vaccines are typically administered via i.m. injection to humans and are incapable of inducing respiratory mucosal immunity. However, aerosol delivery of Ad-vectored vaccines remains poorly characterized, and its ability to induce mucosal immunity in humans is unknown. This phase Ib trial evaluated the safety and immunogenicity of human serotype-5 Ad-vectored tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to humans via inhaled aerosol or i.m. injection.MethodsThirty-one healthy, previously BCG-vaccinated adults were enrolled. AdHu5Ag85A was administered by single-dose aerosol using Aeroneb Solo Nebulizer or by i.m. injection. The study consisted of the low-dose (LD) aerosol, high-dose (HD) aerosol, and i.m. groups. The adverse events were assessed at various times after vaccination. Immunogenicity data were collected from the peripheral blood and bronchoalveolar lavage samples at baseline, as well as at select time points after vaccination.ResultsThe nebulized aerosol droplets were < 5.39 µm in size. Both LD and HD of AdHu5Ag85A administered by aerosol inhalation and i.m. injection were safe and well tolerated. Both aerosol doses, particularly LD, but not i.m., vaccination markedly induced airway tissue-resident memory CD4+ and CD8+ T cells of polyfunctionality. While as expected, i.m. vaccination induced Ag85A-specific T cell responses in the blood, the LD aerosol vaccination also elicited such T cells in the blood. Furthermore, the LD aerosol vaccination induced persisting transcriptional changes in alveolar macrophages.ConclusionInhaled aerosol delivery of Ad-vectored vaccine is a safe and superior way to elicit respiratory mucosal immunity. This study warrants further development of aerosol vaccine strategies against respiratory pathogens, including TB and COVID-19.Trial registrationClinicalTrial.gov, NCT02337270.FundingThe Canadian Institutes for Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada funded this work.

Mucosal immune response in BNT162b2 COVID-19 vaccine recipients.

BACKGROUND: Although the BNT162b2 COVID-19 vaccine is known to induce IgG neutralizing antibodies in serum protecting against COVID-19, it has not been studied in detail whether it could generate specific immunity at mucosal sites, which represent the primary route of entry of SARS-CoV-2. METHODS: Samples of serum and saliva of 60 BNT162b2-vaccinated healthcare workers were collected at baseline, two weeks after the first dose and two weeks after the second dose. Anti-S1-protein IgG and IgA total antibodies titres and the presence of neutralizing antibodies against the Receptor Binding Domain in both serum and saliva were measured by quantitative and by competitive ELISA, respectively. FINDINGS: Complete vaccination cycle generates a high serum IgG antibody titre as a single dose in previously infected seropositive individuals. Serum IgA concentration reaches a plateau after a single dose in seropositive individuals and two vaccine doses in seronegative subjects. After the second dose IgA level was higher in seronegative than in seropositive subjects. In saliva, IgG level is almost two orders of magnitude lower than in serum, reaching the highest values after the second dose. IgA concentration remains low and increases significantly only in seropositive individuals after the second dose. Neutralizing antibody titres were much higher in serum than in saliva. INTERPRETATION: The mRNA BNT162b2 vaccination elicits a strong systemic immune response by drastically boosting neutralizing antibodies development in serum, but not in saliva, indicating that at least oral mucosal immunity is poorly activated by this vaccination protocol, thus failing in limiting virus acquisition upon its entry through this route. FUNDING: This work was funded by the Department of Medicine and Surgery, University of Insubria, and partially supported by Fondazione Umberto Veronesi (COVID-19 Insieme per la ricerca di tutti, 2020).

Polysaccharides derived from Chinese medicinal herbs: A promising choice of vaccine adjuvants.

Adjuvants have been used in vaccines for a long time to promote the body's immune response, reducing vaccine dosage and production costs. Although many vaccine adjuvants are developed, the use in human vaccines is limited because of either limited action or side effects. Therefore, the development of new vaccine adjuvants is required. Many studies have found that natural polysaccharides derived from Traditional Chinese medicine (TCM) possess good immune promoting effects and simultaneously improve humoral, cellular and mucosal immunity. Recently polysaccharide adjuvants have attracted much attention in vaccine preparation because of their intrinsic characteristics: immunomodulation, biocompatibility, biodegradability, low toxicity and safety. This review article systematically analysed the literature on polysaccharides possessing vaccine adjuvant activity from TCM plants, such as Astragalus polysaccharide (APS), Rehmannia glutinosa polysaccharide (RGP), Isatis indigotica root polysaccharides (IRPS), etc. and their derivatives. We believe that polysaccharide adjuvants can be used to prepare the vaccines for clinical use provided their mechanisms of action are studied in detail.

Immunogenicity and Protective Potential of Mucosal Vaccine Formulations Based on Conserved Epitopes of Influenza A Viruses Fused to an Innovative Ring Nanoplatform in Mice and Chickens.

Current inactivated vaccines against influenza A viruses (IAV) mainly induce immune responses against highly variable epitopes across strains and are mostly delivered parenterally, limiting the development of an effective mucosal immunity. In this study, we evaluated the potential of intranasal formulations incorporating conserved IAV epitopes, namely the long alpha helix (LAH) of the stalk domain of hemagglutinin and three tandem repeats of the ectodomain of the matrix protein 2 (3M2e), as universal mucosal anti-IAV vaccines in mice and chickens. The IAV epitopes were grafted to nanorings, a novel platform technology for mucosal vaccination formed by the nucleoprotein (N) of the respiratory syncytial virus, in fusion or not with the C-terminal end of the P97 protein (P97c), a recently identified Toll-like receptor 5 agonist. Fusion of LAH to nanorings boosted the generation of LAH-specific systemic and local antibody responses as well as cellular immunity in mice, whereas the carrier effect of nanorings was less pronounced towards 3M2e. Mice vaccinated with chimeric nanorings bearing IAV epitopes in fusion with P97c presented modest LAH- or M2e-specific IgG titers in serum and were unable to generate a mucosal humoral response. In contrast, N-3M2e or N-LAH nanorings admixed with Montanide™ gel (MG) triggered strong specific humoral responses, composed of serum type 1/type 2 IgG and mucosal IgG and IgA, as well as cellular responses dominated by type 1/type 17 cytokine profiles. All mice vaccinated with the [N-3M2e + N-LAH + MG] formulation survived an H1N1 challenge and the combination of both N-3M2e and N-LAH nanorings with MG enhanced the clinical and/or virological protective potential of the preparation in comparison to individual nanorings. Chickens vaccinated parenterally or mucosally with N-LAH and N-3M2e nanorings admixed with Montanide™ adjuvants developed a specific systemic humoral response, which nonetheless failed to confer protection against heterosubtypic challenge with a highly pathogenic H5N8 strain. Thus, while the combination of N-LAH and N-3M2e nanorings with Montanide™ adjuvants shows promise as a universal mucosal anti-IAV vaccine in the mouse model, further experiments have to be conducted to extend its efficacy to poultry.

KDP, a Lactobacilli Product from Kimchi, Enhances Mucosal Immunity by Increasing Secretory IgA in Mice and Exhibits Antimicrobial Activity.

The potential of KDP, a lactic acid bacterial strain of Lactobacillus sakei, to enhance the production of mucosal specific immunoglobulin A (IgA) in mice and thereby enhance gut mucosal immunity was examined. KDP is composed of dead cells isolated from the Korean traditional food kimchi. Female BALB/c mice orally received 0.25 mg KDP once daily for 5 weeks and were co-administrated ovalbumin (OVA) for negative control and cholera toxin for positive control. Mice administered KDP exhibited increased secretory IgA (sIgA) contents in the small intestine, Peyer's patches, serum, colon, and lungs as examined by ELISA. KDP also significantly increased the gene expression of Bcl-6, IL-10, IL-12p40, IL-21, and STAT4. In addition, KDP acted as a potent antioxidant, as indicated by its significant inhibitory effects in the range of 16.5-59.4% for DPPH, nitric oxide, maximum total antioxidant capacity, and maximum reducing power. Finally, KDP exhibited potent antimicrobial activity as evidenced by a significant decrease in the growth of 7 samples of gram-negative and gram-positive bacteria and Candida albicans. KDP's adjuvant effect is shown to be comparable to that of cholera toxin. We conclude that KDP can significantly enhance the intestine's secretory immunity to OVA, as well as act as a potent antioxidant and antimicrobial agent. These results suggest that orally administered KDP should be studied in clinical trials for antigen-specific IgA production.

Supplementation of Alhagi honey polysaccharides contributes to the improvement of the intestinal immunity regulating the structure of intestinal flora in mice.

Alhagi honey polysaccharides (AH), a main active component of Alhagi honey, are known to possess excellent pharmacological activities and have been widely used as dietary supplements in traditional Chinese medicine for thousands of years. This study is aimed to investigate the heath effect of AH on murine intestinal mucosal immune function and composition of the gut microbiome. ICR mice received daily intragastric administration of AH (three dosages, 200 mg kg-1, 400 mg kg-1, and 800 mg kg-1) or saline for 7 consecutive days. Results indicated an improvement in the intestinal barrier function through increases in secretory immunoglobulin A (sIgA) and ß-defensins. Simultaneously, AH also significantly stimulated IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, and TNF-α cytokine secretion as compared to the control samples. Moreover, hematoxylin and eosin staining showed that AH enhanced the number of intraepithelial lymphocytes (IELs) in the small intestine. An obvious increase in the ratio of IgA+ cells of AH-treatment samples in the lamina propria was also detected by immunohistochemical staining. In addition, the CD3+, CD4+ and CD8+ T-cell ratio in mesenteric lymph nodes and Peyer's patches in the AH-treatment was significantly higher than that in the control group. Furthermore, 16S rDNA gene sequencing was used to monitor the dynamic changes in the gut microbiota. The result revealed that AH significantly increased the indexes of Shannon and obviously decreased the indexes of Simpson, suggesting the enhancement of the diversity and richness of the intestinal microbiome. Moreover, AH modulated the gut microbiome via increasing the abundance of probiotics and decreasing the levels of pathogenic bacteria. In summary, these results indicated that AH could be used as a prebiotic to enhance murine intestinal mucosal immunity and to modulate the gut microbiome.

Glycerol Monolaurate Ameliorated Intestinal Barrier and Immunity in Broilers by Regulating Intestinal Inflammation, Antioxidant Balance, and Intestinal Microbiota.

This study was conducted to investigate the impact of glycerol monolaurate (GML) on performance, immunity, intestinal barrier, and cecal microbiota in broiler chicks. A total of 360 one-day-old broilers (Arbor Acres) with an average weight of 45.7 g were randomly allocated to five dietary groups as follows: basal diet and basal diets complemented with 300, 600, 900, or 1200 mg/kg GML. Samples were collected at 7 and 14 days of age. Results revealed that feed intake increased (P < 0.05) after 900 and 1200 mg/kg GML were administered during the entire 14-day experiment period. Dietary GML decreased (P < 0.05) crypt depth and increased the villus height-to-crypt depth ratio of the jejunum. In the serum and jejunum, supplementation with more than 600 mg/kg GML reduced (P < 0.05) interleukin-1ß, tumor necrosis factor-α, and malondialdehyde levels and increased (P < 0.05) the levels of immunoglobulin G, jejunal mucin 2, total antioxidant capacity, and total superoxide dismutase. GML down-regulate (P < 0.05) jejunal interleukin-1ß and interferon-γ expression and increased (P < 0.05) the mRNA level of zonula occludens 1 and occludin. A reduced (P < 0.05) expression of toll-like receptor 4 and nuclear factor kappa-B was shown in GML-treated groups. In addition, GML modulated the composition of the cecal microbiota of the broilers, improved (P < 0.05) microbial diversity, and increased (P < 0.05) the abundance of butyrate-producing bacteria. Spearman's correlation analysis revealed that the genera Barnesiella, Coprobacter, Lachnospiraceae, Faecalibacterium, Bacteroides, Odoriacter, and Parabacteroides were related to inflammation and intestinal integrity. In conclusion, GML ameliorated intestinal morphology and barrier function in broiler chicks probably by regulating intestinal immune and antioxidant balance, as well as intestinal microbiota.

Role of interleukin-6 in antigen-specific mucosal immunoglobulin A induction by cationic liposomes.

The COVID-19 pandemic, caused by a highly virulent and transmissible pathogen, has proven to be devastating to society. Mucosal vaccines that can induce antigen-specific immune responses in both the systemic and mucosal compartments are considered an effective measure to overcome infectious diseases caused by pathogenic microbes. We have recently developed a nasal vaccine system using cationic liposomes composed of 1,2-dioleoyl-3-trimethylammonium-propane and cholesteryl 3ß-N-(dimethylaminoethyl)carbamate in mice. However, the comprehensive molecular mechanism(s), especially the host soluble mediator involved in this process, by which cationic liposomes promote antigen-specific mucosal immune responses, remain to be elucidated. Herein, we show that intranasal administration of cationic liposomes elicited interleukin-6 (IL-6) expression at the site of administration. Additionally, both nasal passages and splenocytes from mice nasally immunized with cationic liposomes plus ovalbumin (OVA) were polarized to produce IL-6 when re-stimulated with OVA in vitro. Furthermore, pretreatment with anti-IL-6R antibody, which blocks the biological activities of IL-6, attenuated the production of OVA-specific nasal immunoglobulin A (IgA) but not OVA-specific serum immunoglobulin G (IgG) responses. In this study, we demonstrated that IL-6, exerted by nasally administered cationic liposomes, plays a crucial role in antigen-specific IgA induction.

Nasal Immunization With Small Molecule Mast Cell Activators Enhance Immunity to Co-Administered Subunit Immunogens.

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, small molecule mast cell activators may be a more cost-efficient adjuvant alternative that is easily synthesized with high purity compared to M7 or C48/80. To identify novel mast cell activating compounds that could be evaluated for mucosal vaccine adjuvant activity, we employed high-throughput screening to assess over 55,000 small molecules for mast cell degranulation activity. Fifteen mast cell activating compounds were down-selected to five compounds based on in vitro immune activation activities including cytokine production and cellular cytotoxicity, synthesis feasibility, and selection for functional diversity. These small molecule mast cell activators were evaluated for in vivo adjuvant activity and induction of protective immunity against West Nile Virus infection in BALB/c mice when combined with West Nile Virus envelope domain III (EDIII) protein in a nasal vaccine. We found that three of the five mast cell activators, ST101036, ST048871, and R529877, evoked high levels of EDIII-specific antibody and conferred comparable levels of protection against WNV challenge. The level of protection provided by these small molecule mast cell activators was comparable to the protection evoked by M7 (67%) but markedly higher than the levels seen with mice immunized with EDIII alone (no adjuvant 33%). Thus, novel small molecule mast cell activators identified by high throughput screening are as efficacious as previously described mast cell activators when used as nasal vaccine adjuvants and represent next-generation mast cell activators for evaluation in mucosal vaccine studies.

In vitro and in vivo immunoregulatory activity of sulfated fucan from the sea cucumber A. leucoprocta.

The in vitro and in vivo immunoregulatory activity of a water-soluble sulfated fucan AL1-1 from the sea cucumber A. leucoprocta was elucidated. In vitro experiments showed that AL1-1 up-regulated immunostimulatory activities in RAW264.7 cells and that it could successfully promote ROS production and phagocytic activity, increase secretion levels of iNOS, and induce the production of considerable amounts of cytokines (TNF-α, IL-6, IL-1ß and IL-12). We found that toll-like receptor 4 (TLR4) was mainly involved in AL1-1 mediated macrophage activation. AL1-1's in vivo immunomodulatory activity on cyclophosphamide (CY)-treated mice was investigated and it was shown that it could strongly enhance Sig A levels, promote the total antioxidant capacity (T-AOC), and reduce malondialdehyde (MDA) level in the intestine. It could also increase activities of superoxidase dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX). These results demonstrate that AL1-1 has a significant effect on enhancing in vivo and in vitro immune response.

A Gut Reaction to SIV and SHIV Infection: Lower Dysregulation of Mucosal T Cells during Acute Infection Is Associated with Greater Viral Suppression during cART.

Selection of a pre-clinical non-human primate (NHP) model is essential when evaluating therapeutic vaccine and treatment strategies for HIV. SIV and SHIV-infected NHPs exhibit a range of viral burdens, pathologies, and responses to combinatorial antiretroviral therapy (cART) regimens and the choice of the NHP model for AIDS could influence outcomes in studies investigating interventions. Previously, in rhesus macaques (RMs) we showed that maintenance of mucosal Th17/Treg homeostasis during SIV infection correlated with a better virological response to cART. Here, in RMs we compared viral kinetics and dysregulation of gut homeostasis, defined by T cell subset disruption, during highly pathogenic SIVΔB670 compared to SHIV-1157ipd3N4 infection. SHIV infection resulted in lower acute viremia and less disruption to gut CD4 T-cell homeostasis. Additionally, 24/24 SHIV-infected versus 10/19 SIV-infected animals had sustained viral suppression <100 copies/mL of plasma after 5 months of cART. Significantly, the more profound viral suppression during cART in a subset of SIV and all SHIV-infected RMs corresponded with less gut immune dysregulation during acute SIV/SHIV infection, defined by maintenance of the Th17/Treg ratio. These results highlight significant differences in viral control during cART and gut dysregulation in NHP AIDS models and suggest that selection of a model may impact the evaluation of candidate therapeutic interventions for HIV treatment and cure strategies.

Inhibition of elastase enhances the adjuvanticity of alum and promotes anti-SARS-CoV-2 systemic and mucosal immunity.

Alum, used as an adjuvant in injected vaccines, promotes T helper 2 (Th2) and serum antibody (Ab) responses. However, it fails to induce secretory immunoglobulin (Ig) A (SIgA) in mucosal tissues and is poor in inducing Th1 and cell-mediated immunity. Alum stimulates interleukin 1 (IL-1) and the recruitment of myeloid cells, including neutrophils. We investigated whether neutrophil elastase regulates the adjuvanticity of alum, and whether a strategy targeting neutrophil elastase could improve responses to injected vaccines. Mice coadministered a pharmacological inhibitor of elastase, or lacking elastase, developed high-affinity serum IgG and IgA antibodies after immunization with alum-adsorbed protein vaccines, including the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). These mice also developed broader antigen-specific CD4+ T cell responses, including high Th1 and T follicular helper (Tfh) responses. Interestingly, in the absence of elastase activity, mucosal SIgA responses were induced after systemic immunization with alum as adjuvant. Importantly, lack or suppression of elastase activity enhanced the magnitude of anti-SARS-CoV-2 spike subunit 1 (S1) antibodies, and these antibodies reacted with the same epitopes of spike 1 protein as sera from COVID-19 patients. Therefore, suppression of neutrophil elastase could represent an attractive strategy for improving the efficacy of alum-based injected vaccines for the induction of broad immunity, including mucosal immunity.

Temporal Dynamics of T Helper Populations in the Proximal Small Intestine after Oral Bovine Lactoferrin Administration in BALB/c Mice.

Bovine lactoferrin (bLf), a component of milk and a dietary supplement, modulates intestinal immunity at effector and inductor sites. Considering the regional difference in intestinal compartments and the dynamics of local cytokine-producing cells in the gut across time, the aim of this work was to characterize the effects of bLf on the proximal small intestine in a BALB/c murine model of oral administration. Male BALB/c mice were treated with oral bLf vs. saline control as mock by buccal deposition for 28 days. Intestinal secretions were obtained at different time points and cells were isolated from Peyer's patches (PP) and lamina propria (LP) of the proximal small intestine as representative inductor and effector sites, respectively. Total and specific anti-bLF IgA and IgM were determined by enzyme-immuno assay; the percentages of IgA+ and IgM+ plasma cells (PC) and cytokine-producing CD4+ T cells of PP and LP were analyzed by flow cytometry. We found that total and bLf-specific IgA and IgM levels were increased in the intestinal secretions of the bLf group in comparison to mock group and day 0. LP IgA+ PC and IgM+ PC presented an initial elevation on day 7 and day 21, respectively, followed by a decrease on day 28 in comparison to mock. Higher percentages of CD4+ T cells in LP were found in the bLf group. Cytokines-producing CD4+ T cells populations presented a pattern of increases and decreases in the bLf group in both LP and PP. Transforming growth factor beta (TGF-ß)+ CD4+ T cells showed higher percentages after bLf administration with a marked peak at day 21 in both LP and PP in comparison to mock-treated mice. Oral bLf exhibits complex immune properties in the proximal small intestine, where temporal monitoring of the inductor and effector compartments reveals patterns of rises and falls of different cell populations. Exceptionally, TGF-ß+ CD4+ T cells show consistent higher numbers after bLf intervention across time. Our work suggests that isolated measurements do not show the complete picture of the modulatory effects of oral bLf in immunological sites as dynamic as the proximal small intestine.

Cooperation Between Systemic IgG1 and Mucosal Dimeric IgA2 Monoclonal Anti-HIV Env Antibodies: Passive Immunization Protects Indian Rhesus Macaques Against Mucosal SHIV Challenges.

Understanding the interplay between systemic and mucosal anti-HIV antibodies can provide important insights to develop new prevention strategies. We used passive immunization via systemic and/or mucosal routes to establish cause-and-effect between well-characterized monoclonal antibodies and protection against intrarectal (i.r.) SHIV challenge. In a pilot study, for which we re-used animals previously exposed to SHIV but completely protected from viremia by different classes of anti-HIV neutralizing monoclonal antibodies (mAbs), we made a surprise finding: low-dose intravenous (i.v.) HGN194-IgG1, a human neutralizing mAb against the conserved V3-loop crown, was ineffective when given alone but protected 100% of animals when combined with i.r. applied HGN194-dIgA2 that by itself had only protected 17% of the animals. Here we sought to confirm the unexpected synergy between systemically administered IgG1 and mucosally applied dIgA HGN194 forms using six groups of naïve macaques (n=6/group). Animals received i.v. HGN194-IgG1 alone or combined with i.r.-administered dIgA forms; controls remained untreated. HGN194-IgG1 i.v. doses were given 24 hours before - and all i.r. dIgA doses 30 min before - i.r. exposure to a single high-dose of SHIV-1157ipEL-p. All controls became viremic. Among passively immunized animals, the combination of IgG1+dIgA2 again protected 100% of the animals. In contrast, single-agent i.v. IgG1 protected only one of six animals (17%) - consistent with our pilot data. IgG1 combined with dIgA1 or dIgA1+dIgA2 protected 83% (5/6) of the animals. The dIgA1+dIgA2 combination without the systemically administered dose of IgG1 protected 67% (4/6) of the macaques. We conclude that combining suboptimal antibody defenses at systemic and mucosal levels can yield synergy and completely prevent virus acquisition.