Mucosal Immune Responses Research Articles

PSIII-15 Effect of dietary fatty acids composition on growth performance, fecal consistency, and plasma fatty acid profile of weaned pigs

Abstract Dietary lipids are an important source of energy and fatty acids (FA), which have critical roles in mucosal immune responses, epithelial barrier functions, and overall intestinal development. Therefore, including fat in nursery pig diets can be used as a nutritional strategy to optimize pig growth performance and intestinal health after weaning. This study evaluated the effects of two different dietary fatty acid compositions derived from conventional soybean oil (CSO) and/or high oleic soybean oil (HOSO) supplementation on growth performance, fecal consistency, and plasma FA profile of weaned pigs. A total of 208 weaned pigs (body weights = 4.96 ± 0.8 kg) were used in a randomized complete block design and allotted to 16 pens of 13 pigs each. Experimental diets met NRC (2012) requirements and were provided in a 3-phase nursery feeding program: Phase 1 (d 0-d7), Phase 2 (d 8-d21), and Phase 3 (d 22-d42). Pens were assigned to one of two dietary treatments consisting of two dietary FA compositions derived from the supplementation of 3% of either CSO (20% Oleic acid; 50% Linoleic acid; 6% Linolenic acid) or HOSO (44% Oleic acid; 30% Linoleic acid; 3% Linolenic acid). Feed disappearance and body weight were measured at d 0, 21, and 42. Pen fecal score was assigned daily from d 0 to 21 and then 3 times per week until d 42. Blood samples were collected on d 21 (2 pigs/pen) for plasma FA profile analysis. Performance data were analyzed using T-TEST and fecal score using PROC FREQ procedures of SAS. From d 0 to 21, pigs fed CSO supplemented diets had a greater (P ≤ 0.05) body weight (8.6 vs. 7.4 kg) and daily gain (0.17 vs. 0.12 kg). In addition, daily feed intake and feed efficiency tended to be 23% and 16% greater in CSO-fed pigs, respectively. From d 21 to d 42, there were no differences between dietary treatments for growth performance. Overall nursery period, pigs fed CSO had greater (P ≤ 0.1) daily gain than HOSO pigs (0.34 vs. 0.30 kg). Pigs fed CSO had a greater (P ≤ 0.05) plasma concentration of palmitic, linoleic, and linolenic FA while pigs fed HOSO had a greater (P ≤ 0.05) plasma concentration of palmitoleic and oleic FA. There were no differences in fecal scores in wk 1 and 2. However, pigs fed CSO had less incidence of soft and watery feces (χ2 < 0.1) in wk 3 to 6. In conclusion, a greater dietary content of linoleic and linolenic FA promoted pig growth performance and reduced incidence of diarrhea after weaning compared with greater content of oleic FA.

Intranasal influenza-vectored COVID-19 vaccines confer broad protection against SARS-CoV-2 XBB variants in hamsters

Abstract The XBB.1.5 subvariant has garnered significant attention due to its exceptional immune evasion and transmissibility. Significantly, the evolutionary trajectory of SARS-CoV-2 has shown continual progression, with a recent global shift observed from XBB to BA.2.86, exemplified by the emergence of the predominant JN.1 subvariant. This phenomenon highlights the need for vaccines that can provide broad-spectrum antigenic coverage. In this study, we utilized a NS1-deleted (dNS1) influenza viral vector to engineer an updated live attenuated vectored vaccine called dNS1-XBB-RBD. This vaccine encodes the receptor binding domain (RBD) protein of the XBB.1.5 strain. Our findings demonstrate that the dNS1-XBB-RBD vaccine elicits a similar systemic and mucosal immune response compared to its prototypic form, dNS1-RBD. In hamsters, the dNS1-XBB-RBD vaccine provided robust protection against the SARS-CoV-2 immune-evasive strains XBB.1.9.2.1 and Beta. Remarkably, nasal vaccination with dNS1-RBD, which encodes the ancestor RBD gene, also effectively protected hamsters against both the XBB.1.9.2.1 and Beta strains. These results provide valuable insights about nasal influenza-vectored vaccine and present a promising strategy for the development of a broad-spectrum vaccine against COVID-19 in the future.

The TNFSF12/TWEAK Modulates Colonic Inflammatory Fibroblast Differentiation and Promotes Fibroblast-Monocyte Interactions.

Fibroblasts acquire a proinflammatory phenotype in inflammatory bowel disease, but the factors driving this process and how fibroblasts contribute to mucosal immune responses are incompletely understood. TNF superfamily member 12 (TNFSF12, or TNF-like weak inducer of apoptosis [TWEAK]) has gained interest as a mediator of chronic inflammation. In this study, we explore its role as a driver of inflammatory responses in fibroblasts and its contribution to fibroblast-monocyte interaction using human primary colonic fibroblasts, THP-1 and primary monocytes. Recombinant human TWEAK induced the expression of cytokines, chemokines, and immune receptors in primary colonic fibroblasts. The TWEAK upregulated transcriptome shared 29% homology with a previously published transcriptional profile of inflammatory fibroblasts from ulcerative colitis. TWEAK elevated surface expression of activated fibroblast markers and adhesion molecules (podoplanin [PDPN], ICAM-1, and VCAM-1) and secretion of IL-6, CCL2, and CXCL10. In coculture, fibroblasts induced monocyte adhesion and secretion of CXCL1 and IL-8, and they promoted a CD14high/ICAM-1high phenotype in THP-1 cells, which was enhanced when fibroblasts were prestimulated with TWEAK. Primary monocytes in coculture with TWEAK-treated fibroblasts had altered surface expression of CD16 and triggering receptor expressed on myeloid cells-1 (TREM-1) as well as increased CXCL1 and CXCL10 secretion. Conversely, inhibition of the noncanonical NF-κB pathway on colonic fibroblasts with a NF-κB-inducing kinase small molecule inhibitor impaired their ability to induce a CD14high phenotype on monocytes. Our results indicate that TWEAK promotes an inflammatory fibroblast-monocyte crosstalk that may be amenable for therapeutic intervention.

Next-generation probiotic Bacteroides. dorei: Improving the efficacy of COVID-19 vaccinations

Bacteroides. dorei (B. dorei) is regarded as the “next-generation probiotics”, and it has been demonstrated to fight influenza infection and alter the composition of gut microbiota. The role of the gut microbiota in the immune response to antiviral vaccination has been fully established in animal experiments and clinical studies. However, whether oral B. dorei can affect the immune response to COVID-19 vaccines remains unknown. This study reported that B. dorei enhanced SARS-CoV-2 vaccine efficacy as indicated by elevation in immunoglobulin G antibody and neutralizing antibody titers, mucosal immune response and Th1 immune response, and memory immune responses. Moreover, B. dorei alone could induce maturation of dendritic cells to promote Th1 response, which was crucial for antiviral immunity. These results suggest that B. dorei may potentially be used as an immunoadjuvant or immunopotentiator to enhance immune function, especially in individuals with impaired immune function.

COVID-19 impact in Crohn’s disease patients submitted to autologous hematopoietic stem cell transplantation

BACKGROUND Severe acute respiratory syndrome coronavirus 2 is the virus responsible for coronavirus disease 2019 (COVID-19), a disease that has been blamed for inducing or exacerbating symptoms in patients with autoimmune diseases. Crohn's disease (CD) is an inflammatory bowel disease that affects genetically susceptible patients who develop an abnormal mucosal immune response to the intestinal microbiota. Patients who underwent hematopoietic stem cell transplantation (HSCT) are considered at risk for COVID-19. AIM To describe for the first time the impact of COVID-19 in CD patients who had undergone autologous, non-myeloablative HSCT. METHODS In this descriptive study a series of 19 patients were diagnosed with positive COVID-19. For two patients there were reports of the occurrence of two infectious episodes. Parameters related to HSCT, such as time elapsed since the procedure, vaccination status, CD status before and after infection, and clinical manifestations resulting from COVID-19, were evaluated. RESULTS Among the patients with COVID-19, three, who underwent Auto HSCT less than six months ago, relapsed and one, in addition to the CD symptoms, started to present thyroid impairment with positive anti-TPO. Only one of the patients required hospitalization for five days to treat COVID-19 and remained in CD clinical remission. Nine patients reported late symptoms that may be related to COVID-19. There were no deaths, and a statistical evaluation of the series of COVID-19 patients compared to those who did not present any infectious episode did not identify significant differences regarding the analyzed parameters. CONCLUSION Despite the change in CD status in three patients and the presence of nine patients with late symptoms, we can conclude that there was no significant adverse impact concerning COVID-19 in the evaluated patients who underwent HSCT to treat CD.

A universal recombinant adenovirus type 5 vector-based COVID-19 vaccine

A universal recombinant adenovirus type-5 (Ad5) vaccine against COVID19 (Ad-US) was constructed, and immunogenicity and broad-spectrum of Ad5-US were evaluated with both intranasal and intramuscular immunization routes. The humoral immune response of Ad5-US in serum and bronchoalveolar lavage fluid were evaluated by the enzyme-linked immunosorbent assay (ELISA), recombinant vesicular stomatitis virus based pseudovirus neutralization assay, and angiotensin-converting enzyme-2 (ACE2) -binding inhibition assay. The cellular immune response and Th1/Th2 biased immune response of Ad5-US were evaluated by the IFN-γ ELISpot assay, intracellular cytokine staining, and Meso Scale Discovery (MSD) profiling of Th1/Th2 cytokines. Intramuscular priming followed by an intranasal booster with Ad5-US elicited the broad-spectrum and high levels of IgG, IgA, pseudovirus neutralizing antibody (PNAb), and Th1-skewing of the T-cell response. Overall, the adenovirus type-5 vectored universal SARS-CoV-2 vaccine Ad5-US was successfully constructed, and Ad5-US was highly immunogenic and broad spectrum. Intramuscular priming followed by an intranasal booster with Ad5-US induced the high and broad spectrum systemic immune responses and local mucosal immune responses.

Saccharomyces cerevisiae oral immunization in mice using multi-antigen of the African swine fever virus elicits a robust immune response

African swine fever virus (ASFV) is one of the most complex viruses. ASFV is a serious threat to the global swine industry because no commercial vaccines against this virus are currently available except in Vietnam. Moreover, ASFV is highly stable in the environment and can survive in water, feed, and aerosols for a long time. ASFV is transmitted through the digestive and respiratory tract. Mucosal immunity is the first line of defense against ASFV. Saccharomyces cerevisiae (SC), which has been certified by the U.S. Food and Drug Administration and has a generally recognized as safe status in the food industry, was used for oral immunization in this study. ASFV antigens were effectively expressed in recombinant SC strains with high DNA copy numbers and stable growth though surface display technology and chromosome engineering (δ-integration). The recombinant SC strains containing eight ASFV antigens—KP177R, E183L, E199L, CP204L, E248R, EP402R, B602L, and B646L— induced strong humoral and mucosal immune responses in mice. There was no antigenic competition, and these antigens induced Th1 and Th2 cellular immune responses. Therefore, the oral immunization strategy using recombinant SC strains containing multiple ASFV antigens demonstrate potential for future testing in swine, including challenge studies to evaluate its efficacy as a vaccine against ASFV.

A chimeric adenovirus-vectored vaccine based on Beta spike and Delta RBD confers a broad-spectrum neutralization against Omicron-included SARS-CoV-2 variants.

Urgent research into innovative severe acute respiratory coronavirus-2 (SARS-CoV-2) vaccines that may successfully prevent various emerging emerged variants, particularly the Omicron variant and its subvariants, is necessary. Here, we designed a chimeric adenovirus-vectored vaccine named Ad5-Beta/Delta. This vaccine was created by incorporating the receptor-binding domain from the Delta variant, which has the L452R and T478K mutations, into the complete spike protein of the Beta variant. Both intramuscular (IM) and intranasal (IN) vaccination with Ad5-Beta/Deta vaccine induced robust broad-spectrum neutralization against Omicron BA.5-included variants. IN immunization with Ad5-Beta/Delta vaccine exhibited superior mucosal immunity, manifested by higher secretory IgA antibodies and more tissue-resident memory T cells (TRM) in respiratory tract. The combination of IM and IN delivery of the Ad5-Beta/Delta vaccine was capable of synergically eliciting stronger systemic and mucosal immune responses. Furthermore, the Ad5-Beta/Delta vaccination demonstrated more effective boosting implications after two dosages of mRNA or subunit recombinant protein vaccine, indicating its capacity for utilization as a booster shot in the heterologous vaccination. These outcomes quantified Ad5-Beta/Delta vaccine as a favorable vaccine can provide protective immunity versus SARS-CoV-2 pre-Omicron variants of concern and BA.5-included Omicron subvariants.

Polymeric Caffeic Acid Acts as an Antigen Delivery Carrier for Mucosal Vaccine Formulation by Forming a Complex with an Antigenic Protein

The development of mucosal vaccines, which can generate antigen-specific immune responses in both the systemic and mucosal compartments, has been recognized as an effective strategy for combating infectious diseases caused by pathogenic microbes. Our recent research has focused on creating a nasal vaccine system in mice using enzymatically polymerized caffeic acid (pCA). However, we do not yet understand the molecular mechanisms by which pCA stimulates antigen-specific mucosal immune responses. In this study, we hypothesized that pCA might activate mucosal immunity at the site of administration based on our previous findings that pCA possesses immune-activating properties. However, contrary to our initial hypothesis, the intranasal administration of pCA did not enhance the expression of various genes involved in mucosal immune responses, including the enhancement of IgA responses. Therefore, we investigated whether pCA forms a complex with antigenic proteins and enhances antigen delivery to mucosal dendritic cells located in the lamina propria beneath the mucosal epithelial layer. Data from gel filtration chromatography indicated that pCA forms a complex with the antigenic protein ovalbumin (OVA). Furthermore, we examined the promotion of OVA delivery to nasal mucosal dendritic cells (mDCs) after the intranasal administration of pCA in combination with OVA and found that OVA uptake by mDCs was increased. Therefore, the data from gel filtration chromatography and flow cytometry imply that pCA enhances antigen-specific antibody production in both mucosal and systemic compartments by serving as an antigen-delivery vehicle.

Serum and Salivary IgG and IgA Response After COVID-19 Messenger RNA Vaccination

There is still considerable controversy in the literature regarding the capacity of intramuscular messenger RNA (mRNA) vaccination to induce a mucosal immune response. To compare serum and salivary IgG and IgA levels among mRNA-vaccinated individuals with or without previous SARS-CoV-2 infection. In this cohort study, SARS-CoV-2-naive participants and those with previous infection were consecutively included in the CoviCompare P and CoviCompare M mRNA vaccination trials and followed up to day 180 after vaccination with either the BNT162b2 (Pfizer-BioNTech) vaccine or the mRNA-1273 (Moderna) vaccine at the beginning of the COVID-19 vaccination campaign (from February 19 to June 8, 2021) in France. Data were analyzed from October 25, 2022, to July 13, 2023. An ultrasensitive digital enzyme-linked immunosorbent assay was used for the comparison of SARS-CoV-2 spike-specific serum and salivary IgG and IgA levels. Spike-specific secretory IgA level was also quantified at selected times. A total of 427 individuals were included in 3 groups: participants with SARS-CoV-2 prior to vaccination who received 1 single dose of BNT162b2 (Pfizer-BioNTech) (n = 120) and SARS-CoV-2-naive individuals who received 2 doses of mRNA-1273 (Moderna) (n = 172) or 2 doses of BNT162b2 (Pfizer-BioNTech) (n = 135). The median age was 68 (IQR, 39-75) years, and 228 (53.4%) were men. SARS-CoV-2 spike-specific IgG saliva levels increased after 1 or 2 vaccine injections in individuals with previous infection and SARS-CoV-2-naive individuals. After vaccination, SARS-CoV-2-specific saliva IgA levels, normalized with respect to total IgA levels, were significantly higher in participants with previous infection, as compared with the most responsive mRNA-1273 (Moderna) recipients (median normalized levels, 155 × 10-5 vs 37 × 10-5 at day 29; 107 × 10-5 vs 54 × 10-5 at day 57; and 104 × 10-5 vs 70 × 10-5 at day 180 [P < .001]). In contrast, compared with day 1, spike-specific IgA levels in the BNT162b2-vaccinated SARS-CoV-2-naive group increased only at day 57 (36 × 10-5 vs 49 × 10-5 [P = .01]). Bona fide multimeric secretory IgA levels were significantly higher in individuals with previous infection compared with SARS-CoV-2-naive individuals after 2 antigenic stimulations (median optical density, 0.36 [IQR, 0.16-0.63] vs 0.16 [IQR, 0.10-0.22]; P < .001). The findings of this cohort study suggest that mRNA vaccination was associated with mucosal immunity in individuals without prior SARS-CoV-2 infection, but at much lower levels than in previously infected individuals. Further studies are needed to determine the association between specific saliva IgA levels and prevention of infection or transmission.

The Papain-like Protease Domain of Severe Acute Respiratory Syndrome Coronavirus 2 Conjugated with Human Beta-Defensin 2 and Co1 Induces Mucosal and Systemic Immune Responses against the Virus.

Most of the licensed vaccines against SARS-CoV-2 target spike proteins to induce viral neutralizing antibodies. However, currently prevalent SARS-CoV-2 variants contain many mutations, especially in their spike proteins. The development of vaccine antigens with conserved sequences that cross-react with variants of SARS-CoV-2 is needed to effectively defend against SARS-CoV-2 infection. Given that viral infection is initiated in the respiratory mucosa, strengthening the mucosal immune response would provide effective protection. We constructed a mucosal vaccine antigen using the papain-like protease (PLpro) domain of non-structural protein 3 of SARS-CoV-2. To potentiate the mucosal immune response, PLpro was combined with human beta-defensin 2, an antimicrobial peptide with mucosal immune adjuvant activity, and Co1, an M-cell-targeting ligand. Intranasal administration of the recombinant PLpro antigen conjugate into C57BL/6 and hACE2 knock-in (KI) mice induced antigen-specific T-cell and antibody responses with complement-dependent cytotoxic activity. Viral challenge experiments using the Wuhan and Delta strains of SARS-CoV-2 provided further evidence that immunized hACE2 KI mice were protected against viral challenge infections. Our study shows that PLpro is a useful candidate vaccine antigen against SARS-CoV-2 infection and that the inclusion of human beta-defensin 2 and Co1 in the recombinant construct may enhance the efficacy of the vaccine.

Multicentre double-blind randomised placebo-controlled trial evaluating the efficacy of the meningococcal B vaccine, 4CMenB (Bexsero), against Neisseria gonorrhoeae infection in men who have sex with men: the GoGoVax study protocol

IntroductionGonorrhoea, the sexually transmissible infection caused by Neisseria gonorrhoeae, has a substantial impact on sexual and reproductive health globally with an estimated 82 million new infections each year worldwide. N....

Transglutaminase-Cross-Linked Tofu Suppressed Soybean-Induced Allergic Reactions by Enhancing Intestinal Mucosa Immune Tolerance.

Currently, food allergies are closely related to intestinal health, and ensuring the integrity and health of intestinal mucosa could reduce the incidence of food allergies. In this study, a soybean-allergic mouse model was used to explore the mechanism of intestinal mucosa immune response induced by enzyme-cross-linked tofu. The effects of enzyme-cross-linked tofu on intestinal mucosal immunity in mice were determined by hematoxylin-eosin (HE) staining and flow cytometry. Our results reveled that the MTG-cross-linked tofu reduced the reactivity of the intestinal mucosal immune system, which mainly manifested as a decrease in the dendritic cell (DC) levels of mesenteric lymph nodes (MLNs), increasing the Th1 cells and Tregs in Peyer's patch (PP) nodes and MLNs, and inhibiting the Th2 cells. Compared with soy protein, enzyme-cross-linked tofu had less damage to the small intestinal tract of mice. Therefore, the above-mentioned results fully revealed that the enzyme-cross-linked tofu promoted the transformation of intestinal mucosal immune cells, shifted the Th1/Th2 balance toward Th1, and reduced its sensitization effect.

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.

Mucosal Vaccination with a Newcastle Disease Virus-Vectored Vaccine Reduces Viral Loads in SARS-CoV-2-Infected Cynomolgus Macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged following an outbreak of unexplained viral illness in China in late 2019. Since then, it has spread globally causing a pandemic that has resulted in millions of deaths and has had enormous economic and social consequences. The emergence of SARS-CoV-2 saw the rapid and widespread development of a number of vaccine candidates worldwide, and this never-before-seen pace of vaccine development led to several candidates progressing immediately through clinical trials. Many countries have now approved vaccines for emergency use, with large-scale vaccination programs ongoing. Despite these successes, there remains a need for ongoing pre-clinical and clinical development of vaccine candidates against SARS-CoV-2, as well as vaccines that can elicit strong mucosal immune responses. Here, we report on the efficacy of a Newcastle disease virus-vectored vaccine candidate expressing SARS-CoV-2 spike protein (NDV-FLS) administered to cynomolgus macaques. Macaques given two doses of the vaccine via respiratory immunization developed robust immune responses and had reduced viral RNA levels in nasal swabs and in the lower airway. Our data indicate that NDV-FLS administered mucosally provides significant protection against SARS-CoV-2 infection, resulting in reduced viral burden and disease manifestation, and should be considered as a viable candidate for clinical development.

The Trimeric Autotransporter Adhesin SadA from Salmonella spp. as a Novel Bacterial Surface Display System.

Bacterial surface display platforms have been developed for applications such as vaccine delivery and peptide library screening. The type V secretion system is an attractive anchoring motif for the surface expression of foreign proteins in gram-negative bacteria. SadA belongs to subtype C of the type V secretion system derived from Salmonella spp. and promotes biofilm formation and host cell adherence. The inner membrane lipoprotein SadB is important for SadA translocation. In this study, SadA was used as an anchoring motif to expose heterologous proteins in Salmonella typhimurium using SadB. The ability of SadA to display heterologous proteins on the S. typhimurium surface in the presence of SadB was approximately three-fold higher than that in its absence of SadB. Compared to full-length SadA, truncated SadAs (SadA877 and SadA269) showed similar display capacities when exposing the B-cell epitopes of urease B from Helicobacter pylori (UreB158-172aa and UreB349-363aa). We grafted different protein domains, including mScarlet (red fluorescent protein), the urease B fragment (UreBm) from H. pylori SS1, and/or protective antigen domain 4 from Bacillus anthracis A16R (PAD4), onto SadA877 or SadA1292. Whole-cell dot blotting, immunofluorescence, and flow cytometric analyses confirmed the localization of Flag×3-mScarlet (~30 kDa) and Flag×3-UreBm-mScarlet (~58 kDa) to the S. typhimurium surface using truncated SadA877 or SadA1292 as an anchoring motif. However, Flag×3-UreBm-PAD4-mScarlet (~75 kDa) was displayed on S. typhimurium using SadA1292. The oral administrated pSadBA1292-FUM/StmΔygeAΔmurI and pSadBA877-FUM/StmΔygeAΔmurI could elicit a significant mucosal and humoral immunity response. SadA could thus be used as an anchoring motif for the surface expression of large heterologous proteins as a potential strategy for attenuated bacterial vaccine development.

Mapping Human Immunity and the Education of Waldeyer's Ring.

The development and deployment of single-cell genomic technologies have driven a resolution revolution in our understanding of the immune system, providing unprecedented insight into the diversity of immune cells present throughout the body and their function in health and disease. Waldeyer's ring is the collective name for the lymphoid tissue aggregations of the upper aerodigestive tract, comprising the palatine, pharyngeal (adenoids), lingual, and tubal tonsils. These tonsils are the first immune sentinels encountered by ingested and inhaled antigens and are responsible for mounting the first wave of adaptive immune response. An effective mucosal immune response is critical to neutralizing infection in the upper airway and preventing systemic spread, and dysfunctional immune responses can result in ear, nose, and throat pathologies. This review uses Waldeyer's ring to demonstrate how single-cell technologies are being applied to advance our understanding of the immune system and highlight directions for future research.

Mucosal Immunization with Spore-Based Vaccines against Mannheimia haemolytica Enhances Antigen-Specific Immunity.

Mannheimia haemolytica is a bovine respiratory pathogen commonly associated with bacterial bronchopneumonia. Current vaccine strategies have shown variable efficacy in feedlot cattle, and therefore novel vaccines are needed. Bacillus subtilis spores have been investigated as a mucosal vaccine platform, due to their ability to bind and present antigens to the mucosa and act as an adjuvant. The aim of this study was to develop two spore-based mucosal vaccines targeting M. haemolytica and evaluate their immunogenicity in mice. Two antigen constructs composed of cholera toxin B subunit, M. haemolytica leukotoxin, and either the M. haemolytica outer membrane protein PlpE (MhCP1) or GS60 (MhCP2) were synthesized, purified and then bound to spores as vaccines. In two separate mice trials, the spore-bound vaccines (Spore-MhCP1 and Spore-MhCP2) were administered to mice through intranasal and intragastric routes, while free antigens were administered intranasally and intramuscularly. Unbound spores were also evaluated intranasally. Antigen-specific serum IgG and mucosal IgA from bronchoalveolar lavage, feces, and saliva were measured after vaccination. Mice sera from all treatment groups were assessed for their bactericidal activity against M. haemolytica. In both mice experiments, intramuscular immunization induced the strongest serum IgG antibody response. However, the intranasal administration of Spore-MhCP1 and Spore-MhCP2 elicited the greatest secretory IgA-specific response against leukotoxin, PlpE, and GS60 in bronchoalveolar lavage, saliva, and feces (p < 0.05). Compared to the intranasal administration of free antigen, spore-bound antigen groups showed greater bactericidal activity against M. haemolytica (p < 0.05). Since intranasally delivered Spore-MhCP1 and Spore-MhCP2 elicited both systemic and mucosal immune responses in mice, these vaccines may have potential to mitigate lung infection in cattle by restricting M. haemolytica colonization and proliferation in the respiratory tract. The efficacy of these mucosal spore-based vaccines merits further assessment against M. haemolytica in cattle.

Intranasal administration of unadjuvanted SARS-CoV-2 spike antigen boosts antigen-specific immune responses induced by parenteral protein subunit vaccine prime in mice and hamsters.

With the continued transmission of SARS-CoV-2 across widely vaccinated populations, it remains important to develop new vaccines and vaccination strategies capable of providing protective immunity and limiting the spread of disease. Heterologous prime-boost vaccination based on the selection of different vaccine formulations and administration routes for priming and booster doses presents a promising strategy for inducing broader immune responses in key systemic and respiratory mucosal compartments. Intranasal vaccination can induce mucosal immune responses at the site of SARS-CoV-2 infection; however, the lack of clinically approved mucosal adjuvants makes it difficult to induce robust immune responses with protein subunit vaccines. Herein, we evaluated the immunogenicity of heterologous prime-boost regimens in mice and hamsters based on a parenteral vaccination of the antigen in combination with sulfated lactosylarchaeol (SLA) archaeosomes, a liposome adjuvant comprised of a single semisynthetic archaeal lipid, followed by an intranasally administered unadjuvanted SARS-CoV-2 spike antigen. Intranasal administration of unadjuvanted spike to mice and hamsters increased serum spike-specific IgG titers and spike-neutralizing activity compared with nonboosted animals. Spike-specific IgA responses were also detected in the bronchoalveolar lavage fluid in the lungs of mice that received an intranasal boost. In hamsters, the intranasal boost showed high efficacy against SARS-CoV-2 infection by protecting from body weight loss and reducing viral titers in the lungs and nasal turbinate. Overall, our heterologous intramuscular prime-intranasal boost with SLA-adjuvanted and unadjuvanted spike, respectively, demonstrated the potential of protein subunit formulations to promote antigen-specific systemic and mucosal immune responses.

419 Intramuscular immunization with rVCG-MECA vaccine elicits stronger chlamydial specific immune response than intranasal immunization

OBJECTIVES/GOALS: We prioritize Chlamydia’s public health impact, aim to develop rVCG-MECA for practical use, study robust immunity for effective strategies, and assess animal models for human vaccination adaptation. Our work highlights rVCG-MECA’s translational significance in public health. METHODS/STUDY POPULATION: Female Mice C57BL/6J mice (N=8) were immunized intramuscularly(IM) and intranasally(IN) and boosted twice, two weeks apart, with rVCG-MECA, once with live Chlamydia (C. trachomatis serovar D elementary bodies) and PBS. Specific mucosal and systemic immune responses were characterized. Vaccine efficacy was determined from chlamydia shedding following the transcervical challenge. Additionally, Chlamydia-specific cytokine (IFN-γ and IL-4) production by splenic and ILN T cells was assessed after 16 weeks RESULTS/ANTICIPATED RESULTS: Immunization with rVCG-MECA via intramuscular and intranasal routes triggered notable humoral responses in systemic and mucosal tissues. Intramuscular vaccination produced higher IgG2c levels in both tissues, while intranasal vaccination led to elevated IgA levels in mucosal tissues. rVCG-MECA-immunized mice exhibited significantly higher IFN-γ (Th1) secretion compared to IL-4 (Th2), with intramuscular immunization showing the highest IFN-γ levels. These findings anticipate robust immune responses, promising protection against Chlamydia, particularly through the intra muscular route. Overall, our results support rVCG-MECA as a promising Chlamydia vaccine, aligned with public health goals. DISCUSSION/SIGNIFICANCE: This study suggests that IM and IN immunization with rVCG-MECA induces immune effectors such as IFN-gamma and IgG2c that mediate chlamydial clearance in the genetical tract.