Bifidobacterium longum subsp. infantis is a commensal bacterium that predominates in the infant gut, playing a critical role in both preventing foreign infections and facilitating immune development. This study aimed to explore the effects of B. longum subsp. infantis supplementation on interferon-beta (IFN-β) secretion and intestinal barrier improvement in growing mice. Female and male mice were orally administered either saline or B. longum subsp. infantis CCFM1269 or I5TI (1 × 109 CFU/mice per day, n = 8) from 1-week-age until 3-, 4-, and 5-week-age. RNA sequencing analysis revealed that CCFM1269 exhibited potential antiviral capacity through increasing 2’-5’ oligoadenylate synthetase (OAS). Additionally, CCFM1269 supplementation significantly increased colonic IFN-β levels which combined with OAS in 3-week-old female and male mice by activating the TLR4-TRIF-dependent signaling pathway. However, this effect was not observed in 4- and 5-week-old mice. Furthermore, both CCFM1269 were found to modulate the gut microbiota composition and enhance the intestinal barrier function in 3-, 4-, and 5-week-old mice. In summary, the results of this study suggested that B. longum subsp. infantis CCFM1269 promoting intestinal barrier and releasing IFN-β in growing mice was in a strain-specific and time-dependent manner.

Seasonal epidemics of influenza viruses are responsible for a significant global public health burden. Vaccination remains the most effective way to prevent infection; however, due to the persistence of antigenic drift, vaccines must be updated annually. The selection of vaccine strains occurs months in advance of the influenza season to allow adequate time for production in eggs. RNA vaccines offer the potential to accelerate production and improve efficacy of influenza vaccines. We leveraged the nucleoside-modified RNA (modRNA) platform technology and lipid nanoparticle formulation process of the COVID-19 mRNA vaccine (BNT162b2; Comirnaty®) to create modRNA vaccines encoding hemagglutinin (HA) (modRNA-HA) for seasonal human influenza strains and evaluated their preclinical immunogenicity and toxicity. In mice, a monovalent modRNA vaccine encoding an H1 HA demonstrated robust antibody responses, HA-specific Th1-type CD4+ T cell responses, and HA-specific CD8+ T cell responses. In rhesus and cynomolgus macaques, the vaccine exhibited durable functional antibody responses and HA-specific IFN-γ+ CD4+ T cell responses. Immunization of mice with monovalent, trivalent, and quadrivalent modRNA-HA vaccines generated functional antibody responses targeting the seasonal influenza virus(es) encoded in the vaccines that were greater than, or similar to, those of a licensed quadrivalent influenza vaccine. Monovalent and quadrivalent modRNA-HA vaccines were well-tolerated by Wistar Han rats, with no evidence of systemic toxicity. These nonclinical immunogenicity and safety data support further evaluation of the modRNA-HA vaccines in clinical studies.

Abstract IL2 signals pleiotropically on diverse cell types, some of which contribute to therapeutic activity against tumors, whereas others drive undesired activity, such as immunosuppression or toxicity. We explored the theory that targeting of IL2 to CD8+ T cells, which are key antitumor effectors, could enhance its therapeutic index. To this aim, we developed AB248, a CD8 cis-targeted IL2 that demonstrates over 500-fold preference for CD8+ T cells over natural killer and regulatory T cells (Tregs), which may contribute to toxicity and immunosuppression, respectively. AB248 recapitulated IL2’s effects on CD8+ T cells in vitro and induced selective expansion of CD8+T cells in primates. In mice, an AB248 surrogate demonstrated superior antitumor activity and enhanced tolerability as compared with an untargeted IL2Rβγ agonist. Efficacy was associated with the expansion and phenotypic enhancement of tumor-infiltrating CD8+ T cells, including the emergence of a “better effector” population. These data support the potential utility of AB248 in clinical settings. Significance: The full potential of IL2 therapy remains to be unlocked. We demonstrate that toxicity can be decoupled from antitumor activity in preclinical models by limiting IL2 signaling to CD8+ T cells, supporting the development of CD8+ T cell–selective IL2 for the treatment of cancer. See related article by Kaptein et al. p. 1226.

As SARS-CoV-2 evolves, increasing in potential for greater transmissibility and immune escape, updated vaccines are needed to boost adaptive immunity to protect against COVID-19 caused by circulating strains. Here, we report features of the monovalent Omicron XBB.1.5-adapted BNT162b2 vaccine, which contains XBB.1.5-specific sequence changes, relative to the original BNT162b2 backbone, in the encoded prefusion-stabilized SARS-CoV-2 spike protein (S(P2)). Biophysical characterization of Omicron XBB.1.5 S(P2) demonstrated that it maintains a prefusion conformation and adopts a flexible, predominantly open, state, with high affinity for the human ACE-2 receptor. When administered as a 4th dose in BNT162b2-experienced mice, the monovalent Omicron XBB.1.5 vaccine elicited substantially higher serum neutralizing titers against pseudotyped viruses of Omicron XBB.1.5, XBB.1.16, XBB.1.16.1, XBB.2.3, EG.5.1 and HV.1 sublineages and phylogenetically distant BA.2.86 lineage than the bivalent Wild Type + Omicron BA.4/5 vaccine. Similar trends were observed against Omicron XBB sublineage pseudoviruses when the vaccine was administered as a 2-dose series in naive mice. Strong S-specific Th1 CD4+ and IFNγ+ CD8+ T cell responses were also observed. These findings, together with real world performance of the XBB.1.5-adapted vaccine, suggest that preclinical data for the monovalent Omicron XBB.1.5-adapted BNT162b2 was predictive of protective immunity against dominant SARS-CoV-2 strains.

Respiratory syncytial virus (RSV) is the leading, global cause of serious respiratory disease in infants and is an important cause of respiratory illness in older adults. No RSV vaccine is currently available. The RSV fusion (F) glycoprotein is a key antigen for vaccine development, and its prefusion conformation is the target of the most potent neutralizing antibodies. Here, we describe a computational and experimental strategy for designing immunogens that enhance the conformational stability and immunogenicity of RSV prefusion F. We obtained an optimized vaccine antigen after screening nearly 400 engineered F constructs. Through in vitro and in vivo characterization studies, we identified F constructs that are more stable in the prefusion conformation and elicit ~10-fold higher serum-neutralizing titers in cotton rats than DS-Cav1. The stabilizing mutations of the lead construct (847) were introduced onto F glycoprotein backbones of strains representing the dominant circulating genotypes of the two major RSV subgroups, A and B. Immunization of cotton rats with a bivalent vaccine formulation of these antigens conferred complete protection against RSV challenge, with no evidence of disease enhancement. The resulting bivalent RSV prefusion F investigational vaccine has recently been shown to be efficacious against RSV disease in two pivotal phase 3 efficacy trials, one for passive protection of infants by immunization of pregnant women and the second for active protection of older adults by direct immunization.

Antibody analytics and sequences.

Pharmacokinetics of BCMA bispecific and ADC in cynomolgus monkeys.

BCMA cleavable and non-cleavable ADCs are highly active against xenograft tumors.

The development of a vaccine to prevent congenital human cytomegalovirus (HCMV) disease is a public health priority. We tested rhesus CMV (RhCMV) prototypes of HCMV vaccine candidates in a seronegative macaque oral challenge model. Immunogens included a recombinant pentameric complex (PC; gH/gL/pUL128/pUL130/pUL131A), a postfusion gB ectodomain, and a DNA plasmid that encodes pp65-2. Immunization with QS21-adjuvanted PC alone or with the other immunogens elicited neutralizing titers comparable to those elicited by RhCMV infection. Similarly, immunization with all 3 immunogens elicited pp65-specific cytotoxic T-cell responses comparable to those elicited by RhCMV infection. RhCMV readily infected immunized animals and was detected in saliva, blood, and urine after challenge in quantities similar to those in placebo-immunized animals. If HCMV evades vaccine-elicited immunity in humans as RhCMV evaded immunity in macaques, a HCMV vaccine must elicit immunity superior to, or different from, that elicited by the prototype RhCMV vaccine to block horizontal transmission.

Human cytomegalovirus (HCMV) is the leading viral cause of congenital disease and permanent birth defects worldwide. Although the development of an effective vaccine is a public health priority, no vaccines are approved. Among the major antigenic targets are glycoproteins in the virion envelope, including gB, which facilitates cellular entry, and the pentameric complex (gH/gL/pUL128-131), required for the infection of specialized cell types. In this study, sera from rabbits immunized with the recombinant pentameric complex were tested for their ability to neutralize infection of epithelial cells, fibroblasts, and primary placental cell types. Sera from rhesus macaques immunized with recombinant gB or gB plus pentameric complex were tested for HCMV neutralizing activity on both cultured cells and cell column cytotrophoblasts in first-trimester chorionic villus explants. Sera from rabbits immunized with the pentameric complex potently blocked infection by pathogenic viral strains in amniotic epithelial cells and cytotrophoblasts but were less effective in fibroblasts and trophoblast progenitor cells. Sera from rhesus macaques immunized with the pentameric complex and gB more strongly reduced infection in fibroblasts, epithelial cells, and chorionic villus explants than sera from immunization with gB alone. These results suggest that the pentameric complex and gB together elicit antibodies that could have potential as prophylactic vaccine antigens.

Recently, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage called B.1.1.7 (variant of concern: VOC 202012/01), which is reported to spread more efficiently and faster than other strains, emerged in the United Kingdom. This variant has an unusually large number of mutations, with 10 amino acid changes in the spike (S) protein, raising concerns that its recognition by neutralizing antibodies may be affected. In this study, we tested SARS-CoV-2-S pseudoviruses bearing either the Wuhan reference strain or the B.1.1.7 lineage spike protein with sera of 40 participants who were vaccinated in a previously reported trial with the messenger RNA-based COVID-19 vaccine BNT162b2. The immune sera had slightly reduced but overall largely preserved neutralizing titers against the B.1.1.7 lineage pseudovirus. These data indicate that the B.1.1.7 lineage will not escape BNT162b2-mediated protection.

T-cell engaging biologics is a class of novel and promising immune-oncology compounds that leverage the immune system to eradicate cancer. Here, we compared and contrasted a bispecific diabody-Fc format, which displays a relatively short antigen-binding arm distance, with our bispecific IgG platform. By generating diverse panels of antigen-expressing cells where B cell maturation antigen is either tethered to the cell membrane or located to the juxtamembrane region and masked by elongated structural spacer units, we presented a systematic approach to investigate the role of antigen epitope location and molecular formats in immunological synapse formation and cytotoxicity. We demonstrated that diabody-Fc is more potent for antigen epitopes located in the membrane distal region, while bispecific IgG is more efficient for membrane-proximal epitopes. Additionally, we explored other parameters, including receptor density, antigen-binding affinity, and kinetics. Our results show that molecular format and antigen epitope location, which jointly determine the intermembrane distance between target cells and T cells, allow decoupling of cytotoxicity and cytokine release, while antigen-binding affinities appear to be positively correlated with both readouts. Our work offers new insight that could potentially lead to a wider therapeutic window for T-cell engaging biologics in general.

Global warming has led to an increase in the frequency, duration, and intensity of heat waves in the summer, which can cause frequent and acute heat stress on ectotherms. Thus, determining how ectothermic animals respond to heat waves has been attracting growing interest among ecologists. However, the physiological and biochemical responses to heat waves in reptiles, especially aquatic reptiles, are still poorly understood. The current study investigated the oxidant physiology, immunity, and expression levels of heat shock proteins (HSP) mRNA after exposure to a simulated heat wave (1 week, 35 ± 4°C), followed by a recovery period (1 week, 28 ± 4°C) in juvenile Asian yellow pond turtle ( Mauremys mutica ), a widely farmed aquatic turtle in East Asia. The contents of malondialdehyde (MDA) in the liver and muscle were not significantly affected by the heat wave or recovery. Of all antioxidant enzymes, only the activity of glutathione peroxidase (GSH-Px) in muscles increased after heat wave, while the total superoxide dismutase (T-SOD), catalase activity (CAT), and total antioxidant capacity (T-AOC) did not change during the study. The organo-somatic index for the liver and spleen of M. mutica decreased after the heat wave but increased to the initial level after recovery. In contrast, plasma lysozyme activity and serum complement C4 levels increased after the heat wave, returning to the control level after recovery. In addition, heat waves did not alter the relative expression of HSP60, HSP70, and HSP90 mRNA in the liver. Eventually, heat wave slightly increased the IBR/n index. Therefore, our results suggested that heat waves did not lead to oxidative damage to lipids in M. mutica , but deleteriously affected the turtles’ immune organs. Meanwhile, the constitutive levels of most antioxidative enzyme activities, HSPs and enhanced blood immune functions might protect the turtles from the threat of heat waves under the current climate scenarios.

FLT3 (FMS-like tyrosine kinase 3), expressed on the surface of acute myeloid leukemia (AML) blasts, is a promising AML target, given its role in the development and progression of leukemia, and its limited expression in tissues outside the hematopoietic system. Small molecule FLT3 kinase inhibitors have been developed, but despite having clinical efficacy, they are effective only on a subset of patients and associated with high risk of relapse. A durable therapy that can target a wider population of AML patients is needed. Here, we developed an anti-FLT3-CD3 immunoglobulin G (IgG)-based bispecific antibody (7370) with a high affinity for FLT3 and a long half-life, to target FLT3-expressing AML blasts, irrespective of FLT3 mutational status. We demonstrated that 7370 has picomolar potency against AML cell lines in vitro and in vivo. 7370 was also capable of activating T cells from AML patients, redirecting their cytotoxic activity against autologous blasts at low effector-to-target (E:T) ratio. Additionally, under our dosing regimen, 7370 was well tolerated and exhibited potent efficacy in cynomolgus monkeys by inducing complete but reversible depletion of peripheral FLT3(+) dendritic cells (DCs) and bone marrow FLT3(+) stem cells and progenitors. Overall, our results support further clinical development of 7370 to broadly target AML patients.

BACKGROUND:CD40 is a compelling target for cancer immunotherapy, however, attempts to successfully target this pathway have consistently been hampered by dose-limiting toxicity issues in the clinic that prevents the administration of efficacious doses.METHODS:Here, using cytokine and cytokine receptor depletion strategies in conjunction with a potent CD40 agonist, we investigated mechanisms underlying the two primary sources of CD40 agonist-associated toxicity, hepatotoxicity and cytokine release syndrome (CRS).RESULTS:We demonstrate that CD40 agonist -induced hepatotoxicity and CRS are mechanistically independent. Historical data have supported a role for interleukin-6 (IL-6) in CRS-associated wasting, however, our findings instead show that an inflammatory cytokine network involving TNF, IL-12p40, and IFNγ underlie this process. Deficiency of TNF or IFNγ did not influence CD40-induced hepatitis however loss of IL-12p40 significantly decreased circulating concentrations of liver enzymes and reduced the frequency of activated CD14+MHCII+ myeloid cells in the liver, indicating a role for IL-12p40 in liver pathology.CONCLUSIONS:As clinical research programs aim to circumnavigate toxicity concerns while maintaining antitumor efficacy it will be essential to understand which features of CD40 biology mediate antitumor function to develop both safe and efficacious agonists.

Background: Abietic acid (AA) is one of the terpenoids, which are multifunctional natural compounds. It has been reported that AA possesses favorable therapeutic effects on in fl ammation and obesity. Method: In the present study, we determined the inhibitory effect of AA on the proliferation and growth of non-small-cell lung cancer (NSCLC) cell lines for the fi rst time. Then, fl ow cytometry and Western blot analysis were applied to determine the cell apoptosis and cell cycle. Finally, surface plasmon resonance, molecular docking and molecular dynamics (MD) simulation were performed to explore the underlying molecular mechanisms. Results: In vitro experiments indicated that AA displays signi fi cant anti-proliferative, cell cycle arresting and pro-apoptotic activities. Mechanistically, AA abrogated tumor necrosis factor- α induced phosphorylation of I κ B kinase (IKK α / β ) (Ser176/180) and IkB α (Ser32), and inhibited the nuclear translocation of nuclear factor- κ B. Moreover, we found that the activities of AA against NSCLC cells were mediated by its IKK β inhibition. Molecular docking and MD simulations demonstrated that the mechanism of action between AA and IKK β was through hydrophobic interactions. Conclusion: Our data indicate that AA could be a promising lead compound for the discovery of novel IKK β inhibitors and potential agents for the treatment of NSCLC.

The ecophysiological characteristics of native Phragmites australis and exotic Spartina alterniflora grown under waterlogging and salinity were investigated to explore their adaptation potential to sea level rise. The seasonal course of phenotypic traits, photosynthetic activity and chlorophyll fluorescence parameters of P. australis did not change remarkably under shallow flooding, whereas these variables were sensitive to increasing salinity. Waterlogging exacerbated the negative effects of salinity on shoot growth and photosynthetic activity of P. australis, and the combined stresses led to an absence of tassel and reproductive organs. By contrast, S. alterniflora performed well under both stresses and showed an obvious adaptation of salt secretion with increasing salinity. Light salinity was the optimal condition for S. alterniflora, and the tassel growth, chlorophyll content and fluorescence characters under moderate stresses did not differ notably. The Na+ and Cl− concentrations in leaves of both species increased, and the K+ content decreased in response to salinity. Under moderate and high saline levels, the ion concentrations in S. alterniflora were maintained at relatively consistent levels with increased salt secretion. We expect the degradation of P. australis and further colonization of S. alterniflora under prolonged flooding and saltwater intrusion from sea level rise on the coastline of China.