Identification of an Optimized Receptor-Binding Domain Subunit Vaccine against SARS-CoV-2.

Current vaccine efforts to combat SARS-CoV-2 are focused on the whole spike protein administered as mRNA, viral vector, or protein subunit. However, the SARS-CoV-2 receptor-binding domain (RBD) is the immunodominant portion of the spike protein, accounting for 90% of serum neutralizing activity. In this study, we constructed several versions of RBD and together with aluminum hydroxide or DDA (dimethyldioctadecylammonium bromide)/TDB (d-(+)-trehalose 6,6'-dibehenate) adjuvant evaluated immunogenicity in mice. We generated human angiotensin-converting enzyme 2 knock-in mice to evaluate vaccine efficacy in vivo following viral challenge. We found that 1) subdomain (SD)1 was essential for the RBD to elicit maximal immunogenicity; 2) RBDSD1 produced in mammalian HEK cells elicited better immunogenicity than did protein produced in insect or yeast cells; 3) RBDSD1 combined with the CD4 Th1 adjuvant DDA/TDB produced higher neutralizing Ab responses and stronger CD4 T cell responses than did aluminum hydroxide; 4) addition of monomeric human Fc receptor to RBDSD1 (RBDSD1Fc) significantly enhanced immunogenicity and neutralizing Ab titers; 5) the Beta version of RBDSD1Fc provided a broad range of cross-neutralization to multiple antigenic variants of concern, including Omicron; and 6) the Beta version of RBDSD1Fc with DDA/TDB provided complete protection against virus challenge in the knock-in mouse model. Thus, we have identified an optimized RBD-based subunit vaccine suitable for clinical trials.

Neutralizing Antibody Responses to Chlamydia trachomatis in Women and Associations with Chlamydia Outcomes.

We assessed neutralizing antibody responses in a well-characterized cohort of 60 women with different Chlamydia trachomatis infection outcomes noted at a treatment visit and 3-month follow-up. We found varying rates of neutralization (inhibition of C. trachomatis) in sera at different dilution levels and varying neutralizing antibody titers across outcomes. Median neutralization rates were significantly higher in sera at high dilutions (1:320-1:1280) from women with spontaneous resolution versus persisting infection before treatment (all P< .05). These findings suggest that neutralizing antibody responses may contribute to protective immunity against chlamydia.

Dietary Lipid Type, Rather Than Total Number of Calories, Alters Outcomes of Enteric Infection in Mice.

Dietary lipids modulate immunity, yet the means by which specific fatty acids affect infectious disease susceptibility remains unclear. Deciphering lipid-induced immunity is critical to understanding the balance required for protecting against pathogens while avoiding chronic inflammatory diseases. To understand how specific lipids alter susceptibility to enteric infection, we fed mice isocaloric, high-fat diets composed of corn oil (rich in n-6 polyunsaturated fatty acids [n-6 PUFAs]), olive oil (rich in monounsaturated fatty acids), or milk fat (rich in saturated fatty acids) with or without fish oil (rich in n-3 PUFAs). After 5 weeks of dietary intervention, mice were challenged with Citrobacter rodentium, and pathological responses were assessed. Olive oil diets resulted in little colonic pathology associated with intestinal alkaline phosphatase, a mucosal defense factor that detoxifies lipopolysaccharide. In contrast, while both corn oil and milk fat diets resulted in inflammation-induced colonic damage, only milk fat induced compensatory protective responses, including short chain fatty acid production. Fish oil combined with milk fat, unlike unsaturated lipid diets, had a protective effect associated with intestinal alkaline phosphatase activity. Overall, these results reveal that dietary lipid type, independent of the total number of calories associated with the dietary lipid, influences the susceptibility to enteric damage and the benefits of fish oil during infection.

Antibody responses to Chlamydia trachomatis vaccine candidate antigens in Chlamydia-infected women and correlation with antibody-mediated phagocytosis of elementary bodies.

Murine research has revealed a significant role for antibody responses in protection against Chlamydia reinfection. To explore potential humoral immune markers of protection elicited by Chlamydia trachomatis (CT) antigens in humans in the context of presumed clinical correlates of protection, we used both an IgG1-based ELISA and a conventional total IgG ELISA to evaluate antibody responses. We evaluated responses to five CT outer membrane proteins (PmpE, PmpF, PmpG, PmpH, and MOMP), along with other promising CT antigens (Pgp3 and HSP60), negative control antigens (RecO and AtpE), and CT elementary bodies (EBs) in sera from a well-characterized cohort of 60 women with different CT infection outcomes, including two outcomes that are likely clinical correlates of protective immunity: spontaneous resolution of infection and absence of reinfection after treatment. Furthermore, we used a flow cytometry-based assay to measure antibody-mediated phagocytosis by neutrophils in these sera. Results demonstrated that IgG1 ELISA displayed higher sensitivity than conventional total IgG ELISA in assessing antibody responses to CT EBs and antigens. Pgp3 IgG1 ELISA exhibited the highest sensitivity compared to IgG1 ELISA incorporating CT EBs or other antigens, confirming Pgp3 IgG1 ELISA as an ideal assay for CT antibody detection. Most (95%) sera from women with CT infection outcomes exhibited antibody-mediated phagocytosis of CT EBs, which was significantly correlated with IgG1 antibody responses to MOMP, Pgp3, HSP60, and PmpF. However, neither IgG1 responses to CT antigens and EBs nor antibody-mediated phagocytosis were associated with clinical correlates of protection. These findings suggest that neither CT IgG1 antibody detection nor antibody-mediated phagocytosis will be useful as immune correlates of protection against CT infection in humans.

Early life environmental exposures have a minor impact on the gut ecosystem following a natural birth.

A growing body of evidence suggests that the environment is an important source of colonizing bacteria for the gastrointestinal tract of C-section delivered infants, who undergo multiple birth-related interventions; however, the extent to which environmental microbes impact vaginally delivered infants remains unclear. Here we investigated the impact of rural and urban environmental exposures on microbial establishment and immunity in vaginally delivered mice. We simulated rural and urban home environments by adding soil types to cages from breeding to weaning. Our aims were to determine the impact of rural and urban soil exposures on the gut microbiome in young mice and to understand whether these changes persisted into adulthood. Host immune cytokines and microbial short-chain fatty acids were quantified to understand the impact on immunity. We found that early-life soil exposure had a minor effect on the richness of the neonatal gut microbiota contributing 5% and 9% variation in the bacterial community structure between mice during early-life and adulthood, respectively. Exposure to urban soil increased Clostridiaceae and propionic acid which persisted into adulthood. While soil exposure had a limited effect on the gut taxa, systemic cytokine and chemokine profiles were altered in adulthood. The findings presented here show that unlike in C-section deliveries previously reported, environmental exposures following a natural birth have a limited impact on the gut microbial taxa but potentially play an important role in immune-mediated disease susceptibility later in life.

Maternal Intake of Dietary Fat Pre-Programs Offspring's Gut Ecosystem Altering Colonization Resistance and Immunity to Infectious Colitis in Mice.

SCOPE: The transgenerational impact of dietary fat remains unclear. Here, the role of maternal fat consumption as a modulator of gut microbial communities and infectious disease outcomes in their offspring is explored. METHODS AND RESULTS: C57BL/6 mice are fed isocaloric high-fat diets throughout breeding, gestation and lactation. Diets contained either milk fat (MF), olive oil (OO) or corn oil (CO), with or without fish oil. The pups born to maternally exposed mice are weaned on to chow and raised into adulthood. At 8 weeks, the offsprings are either euthanized for colonic 16S rRNA analysis or challenged with the enteric pathogen, Citrobacter rodentium. Maternal CO exposure resulted in unique clustering of bacterial communities in offspring compared with MF and OO. Diets rich in CO reduced survival in offspring challenged with C. rodentium. The addition of fish oil did not improve mortality caused by CO and worsened disease outcomes when combined with OO. Unlike the unsaturated diets, MF is protective with and without fish oil. CONCLUSIONS: Overall, these data reveal that maternal intake of fatty acids do have transgenerational impacts on their offspring's bacteriome and enteric infection risk. Based on this study, saturated fats should be included in maternal diets.

Perinatal lipid nutrition alters early intestinal development and programs the response to experimental colitis in young adult rats.

The long-chain polyunsaturated n-6 and n-3 fatty acids are essential nutrients in membrane biogenesis and regulate gene expression via their eicosanoid metabolites. We investigated whether the n-6 and n-3 fatty acid supply as determined by maternal diet alters colonic phospholipid fatty acids, intestinal morphology, and epithelial barrier permeability during milk feeding with lasting effect on mucosal responsiveness to dinitrobenzene sulfonic acid (DNBS)-induced colitis in young adulthood. Female rats were fed diets with 20% energy from safflower oil (SO) or canola oil (CO), or 8% fish oil (FO) plus 2% SO (10% FO) or 18% FO plus 2% SO (20% FO) throughout gestation and lactation and offspring weaned to a standard diet at 21 days of age. At 15 days of age, pups in the 20% and 10% FO groups had lower 20:4n-6 and higher 20:5n-3 and 22:6n-3 in colon phospholipids (P < 0.01), shorter crypts (P < 0.05), and higher paracellular permeability than SO or CO groups. At 3 mo of age, male offspring in the FO groups showed lasting reduction of crypt depth and a heightened inflammatory response to DNBS. We demonstrate that early decreased colon 20:4n-6 with increased n-3 fatty acids impairs intestinal barrier development and sensitizes the colon response to inflammatory insults later in life.

A high-fat diet rich in corn oil reduces spontaneous locomotor activity and induces insulin resistance in mice.

Over the last few decades, polyunsaturated fatty acid (PUFA), especially n-6 PUFA, and monounsaturated fatty acid content in 'Western diets' has increased manyfold. Such a dietary shift also parallels rising sedentary behavior and diabetes in the Western world. We queried if a shift in dietary fats could be linked to physical inactivity and insulin insensitivity in mice. Eight-week old female C57/Bl6 mice were fed either high-fat (HF) diets [40% energy corn oil (CO) or isocaloric olive oil (OO) diets] or chow (n=10/group) for 6 weeks, followed by estimation of spontaneous locomotor activity, body composition and in vivo metabolic outcomes. Although lean mass and resting energy expenditure stayed similar in both OO- and CO-fed mice, only CO-fed mice demonstrated reduced spontaneous locomotor activity. Such depressed activity in CO-fed mice was accompanied by a lower respiratory ratio, hyperinsulinemia and impaired glucose disposal following intraperitoneal glucose tolerance and insulin tolerance tests compared to OO-fed mice. Unlike the liver, where both HF diets increased expression of fat oxidation genes like PPARs, the skeletal muscle of CO-fed mice failed to up-regulate such genes, thereby supporting the metabolic insufficiencies observed in these mice. In summary, this study demonstrates a specific contribution of n-6 PUFA-rich oils like CO to the loss of spontaneous physical activity and insulin sensitivity in mice. If these data hold true for humans, this study could provide a novel link between recent increases in dietary n-6 PUFA to sedentary behavior and the development of insulin resistance in the Western world.

DNBS/TNBS colitis models: providing insights into inflammatory bowel disease and effects of dietary fat.

Inflammatory Bowel Diseases (IBD), including Crohn's Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48 hr and rats fed SO by 72 hr. Weight gain resumed after 72 hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5 days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

Fish oil attenuates omega-6 polyunsaturated fatty acid-induced dysbiosis and infectious colitis but impairs LPS dephosphorylation activity causing sepsis.

Clinically, excessive ω-6 polyunsaturated fatty acid (PUFA) and inadequate ω-3 PUFA have been associated with enhanced risks for developing ulcerative colitis. In rodent models, ω-3 PUFAs have been shown to either attenuate or exacerbate colitis in different studies. We hypothesized that a high ω-6: ω-3 PUFA ratio would increase colitis susceptibility through the microbe-immunity nexus. To address this, we fed post-weaned mice diets rich in ω-6 PUFA (corn oil) and diets supplemented with ω-3 PUFA (corn oil+fish oil) for 5 weeks. We evaluated the intestinal microbiota, induced colitis with Citrobacter rodentium and followed disease progression. We found that ω-6 PUFA enriched the microbiota with Enterobacteriaceae, Segmented Filamentous Bacteria and Clostridia spp., all known to induce inflammation. During infection-induced colitis, ω-6 PUFA fed mice had exacerbated intestinal damage, immune cell infiltration, prostaglandin E2 expression and C. rodentium translocation across the intestinal mucosae. Addition of ω-3 PUFA on a high ω-6 PUFA diet, reversed inflammatory-inducing microbial blooms and enriched beneficial microbes like Lactobacillus and Bifidobacteria, reduced immune cell infiltration and impaired cytokine/chemokine induction during infection. While, ω-3 PUFA supplementation protected against severe colitis, these mice suffered greater mortality associated with sepsis-related serum factors such as LPS binding protein, IL-15 and TNF-α. These mice also demonstrated decreased expression of intestinal alkaline phosphatase and an inability to dephosphorylate LPS. Thus, the colonic microbiota is altered differentially through varying PUFA composition, conferring altered susceptibility to colitis. Overall, ω-6 PUFA enriches pro-inflammatory microbes and augments colitis; but prevents infection-induced systemic inflammation. In contrast, ω-3 PUFA supplementation reverses the effects of the ω-6 PUFA diet but impairs infection-induced responses resulting in sepsis. We conclude that as an anti-inflammatory agent, ω-3 PUFA supplementation during infection may prove detrimental when host inflammatory responses are critical for survival.

Viral evolution in macaques coinfected with CCR5- and CXCR4-tropic SHIVs in the presence or absence of vaccine-elicited anti-CCR5 SHIV neutralizing antibodies.

Macaques were immunized with SF162 Env-based gp140 immunogens and challenged simultaneously with the CCR5-tropic homologous SHIV(SF162P4) and the CXCR4-tropic heterologous SHIV(SF33A) viruses. Both mock-immunized and immunized animals became dually infected. Prior immunization preferentially reduced the viral replication of the homologous virus during primary infection but the relative replication of the two coinfecting viruses during chronic infection was unaffected by prior immunization, despite the fact that five of six immunized animals maintained a significantly lower overall viral replication that the control animals. Neutralizing antibodies participated in controlling the replication of SHIV(SF162P4), but not that of SHIV(SF33A). Dual infection resulted in the emergence and predominance within the circulating CCR5 virus pool, of a variant with a distinct neutralization phenotype. The signature of this variant was the presence of three amino acid changes in gp120, two of which were located in the receptor and coreceptor binding sites. Also, a significant fraction of the viruses circulating in the blood, as early as two weeks post-infection, was recombinants and prior immunization did not prevent their emergence. These findings provide new insights into the dynamic interaction of CCR5- and CXCR4-tropic HIV isolates that are potentially relevant in better understanding HIV-mediated pathogenesis.

[Expression and immunogenicity of equine infectious anemia virus membrane protein GP90].

BACKGROUND: Membrane protein GP90 of China equine infectious anemia virus (EIAV) vaccine strain (DLV) and its parental wild type LN strain were expressed with Bac-to-Bac baculovirus expression system and BALB/c mice were inoculated with purified protein, thereby to explore the availability of protein for differential diagnosis and potential for preparing genetically engineered vaccine. METHODS: The authors infected donkey PBMC culture with China EIAV vaccine strain (DLV) and its parental wild type LN strain, extracted its proviral DNA as template, amplified the GP90 of DLV and LN, respectively, and expressed with Bac-to-Bac baculovirus expression system. The sf9 insect cells were infected with the recombinant baculovirus and the expressed proteins were purified by IMAC. BALB/c mice were inoculated with purified protein. The specific binding Abs generated in the immunized mice were determined by ELISA method. The neutralizing assay was set up to determine the neutralizing capability of the antigens generated in immunized animals. RESULTS: The recombinant virus expressing viral antigens was determined by Western blot. The expressed proteins were purified by IMAC resulting in the protein purity of 87%(DLV) and 82%(LN), respectively. The antibody titer of the groups with and without adjuvant was 1 600 and 800, respectively. Serial 2 fold dilutions of the immunized mice sera were reacted with 100 TCID50 of EIAV. The end point of immunization assay was to protect 50% cells form CPE caused by EIAV in donkey skin cells. The neutralizing antibody titer was in the range 40 to 80 from animal immunized with and without adjuvant. CONCLUSIONS: Membrane proteins of EIAV vaccine strain and wild type strain were successfully expressed in eukaryotic cell expression system according to the scheduled plan. The proteins showed strong immunogenicity and could activate animals to produce anti-EIAV specific antibody including neutralizing antibody to EIAV.