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Tumor antigen-specific CD4 + T cells generally orchestrate and regulate immune cells to provide immune surveillance against malignancy. However, activation of antigen-specific CD4 + T cells is restricted at local tumor sites where antigen-presenting cells (APCs) are frequently dysfunctional, which can cause rapid exhaustion of anti-tumor immune responses. Herein, we characterize anti-tumor effects of a unique human CD4 + helper T-cell subset that directly recognizes the cytoplasmic tumor antigen, NY-ESO-1, presented by MHC class II on cancer cells. Upon direct recognition of cancer cells, tumor-recognizing CD4 + T cells (TR-CD4) potently induced IFN-γ-dependent growth arrest in cancer cells. In addition, direct recognition of cancer cells triggers TR-CD4 to provide help to NY-ESO-1-specific CD8 + T cells by enhancing cytotoxic activity and improving viability and proliferation in the absence of APCs. Notably, the TR-CD4 either alone or in collaboration with CD8 + T cells significantly inhibited tumor growth in vivo in a xenograft model. Finally, retroviral gene-engineering with T cell receptor (TCR) derived from TR-CD4 produced large numbers of functional TR-CD4. These observations provide mechanistic insights into the role of TR-CD4 in tumor immunity and suggest that approaches to utilize TR-CD4 will augment anti-tumor immune responses for durable therapeutic efficacy in cancer patients.

Interferons (IFNs) are a group of cytokines with a well-established antiviral function. They can be induced by viral infection, are secreted and bind to specific receptors on the same or neighbouring cells to activate the expression of hundreds of IFN stimulated genes (ISGs) with antiviral function. Type I IFN has been known for more than half a century. However, more recently, type III IFN (IFNλ, IL-28/29) was shown to play a similar role and to be particularly important at epithelial surfaces. Here we show that airway epithelia, the primary target of influenza A virus, produce both IFN I and III upon infection, and that induction of both depends on the RIG-I/MAVS pathway. While IRF3 is generally regarded as the transcription factor required for initiation of IFN transcription and the so-called \"priming loop\", we find that IRF3 deficiency has little impact on IFN expression. In contrast, lack of IRF7 reduced IFN production significantly, and only IRF3(-/-)IRF7(-/-) double deficiency completely abolished it. The transcriptional response to influenza infection was largely dependent on IFNs, as it was reduced to a few upregulated genes in epithelia lacking receptors for both type I and III IFN (IFNAR1(-/-)IL-28Rα(-/-)). Wild-type epithelia and epithelia deficient in either the type I IFN receptor or the type III IFN receptor exhibit similar transcriptional profiles in response to virus, indicating that none of the induced genes depends selectively on only one IFN system. In chimeric mice, the lack of both IFN I and III signalling in the stromal compartment alone significantly increased the susceptibility to influenza infection. In conclusion, virus infection of airway epithelia induces, via a RIG-I/MAVS/IRF7 dependent pathway, both type I and III IFNs which drive two completely overlapping and redundant amplification loops to upregulate ISGs and protect from influenza infection.

Gonorrhea, a sexually transmitted disease, causes substantial global morbidity and economic burden. New prevention and control measures for this disease are urgently needed, as strains resistant to almost all classes of antibiotics available for treatment have emerged. Previous reports demonstrate that cross-protection from gonococcal infections may be conferred by meningococcal serogroup B (MenB) outer membrane vesicle (OMV)-based vaccines. Among 1,525 common proteins shared across the genomes of both N. gonorrhoeae and N. meningitidis , 57 proteins were predicted to be surface expressed (outer membrane proteins [OMPs]) and thus preferred targets for vaccine development. The majority of these OMPs showed high sequence identity between the 2 bacterial species. Our results provide valuable insight into the meningococcal antigens present in the current OMV-containing MenB-4C vaccine that may contribute to cross-protection against gonorrhea and may inform next steps in gonorrhea vaccine development. The human pathogens Neisseria gonorrhoeae and Neisseria meningitidis share high genome identity. Retrospective analysis of surveillance data from New Zealand indicates the potential cross-protective effect of outer membrane vesicle (OMV) meningococcal serogroup B vaccine (MeNZB) against N. gonorrhoeae . A licensed OMV-based MenB vaccine, MenB-4C, consists of a recombinant FHbp, NhbA, NadA, and the MeNZB OMV. Previous work has identified several abundantly expressed outer membrane proteins (OMPs) as major components of the MenB-4C OMV with high sequence similarity between N. gonorrhoeae and N. meningitidis , suggesting a mechanism for cross-protection. To build off these findings, we performed comparative genomic analysis on 970 recent N. gonorrhoeae isolates collected through a U.S surveillance system against N. meningitidis serogroup B (NmB) reference sequences. We identified 1,525 proteins that were common to both Neisseria species, of which 57 proteins were predicted to be OMPs using in silico methods. Among the MenB-4C antigens, NhbA showed moderate sequence identity (73%) to the respective gonococcal homolog, was highly conserved within N. gonorrhoeae , and was predicted to be surface expressed. In contrast, the gonococcal FHbp was predicted not to be surface expressed, while NadA was absent in all N. gonorrhoeae isolates. Our work confirmed recent observations (E. A. Semchenko, A. Tan, R. Borrow, and K. L. Seib, Clin Infect Dis, 2018, https://doi.org/10.1093/cid/ciy1061 ) and describes homologous OMPs from a large panel of epidemiologically relevant N. gonorrhoeae strains in the United States against NmB reference strains. Based on our results, we report a set of OMPs that may contribute to the previously observed cross-protection and provide potential antigen targets to guide the next steps in gonorrhea vaccine development. IMPORTANCE Gonorrhea, a sexually transmitted disease, causes substantial global morbidity and economic burden. New prevention and control measures for this disease are urgently needed, as strains resistant to almost all classes of antibiotics available for treatment have emerged. Previous reports demonstrate that cross-protection from gonococcal infections may be conferred by meningococcal serogroup B (MenB) outer membrane vesicle (OMV)-based vaccines. Among 1,525 common proteins shared across the genomes of both N. gonorrhoeae and N. meningitidis , 57 proteins were predicted to be surface expressed (outer membrane proteins [OMPs]) and thus preferred targets for vaccine development. The majority of these OMPs showed high sequence identity between the 2 bacterial species. Our results provide valuable insight into the meningococcal antigens present in the current OMV-containing MenB-4C vaccine that may contribute to cross-protection against gonorrhea and may inform next steps in gonorrhea vaccine development.

Characterizing the immune response to pneumococcal proteins is critical in understanding this bacterium’s epidemiology and vaccinology. Probing a custom-designed proteome microarray with sera from 35 healthy US adults revealed a continuous distribution of IgG affinities for 2,190 potential antigens from the species-wide pangenome. Reproducibly elevated IgG binding was elicited by 208 “antibody binding targets” (ABTs), which included 109 variants of the diverse pneumococcal surface proteins A and C (PspA and PspC) and zinc metalloprotease A and B (ZmpA and ZmpB) proteins. Functional analysis found ABTs were enriched in motifs for secretion and cell surface association, with extensive representation of cell wall synthesis machinery, adhesins, transporter solute-binding proteins, and degradative enzymes. ABTs were associated with stronger evidence for evolving under positive selection, although this varied between functional categories, as did rates of diversification through recombination. Particularly rapid variation was observed at some immunogenic accessory loci, including a phage protein and a phase-variable glycosyltransferase ubiquitous among the diverse set of genomic islands encoding the serine-rich PsrP glycoprotein. Nevertheless, many antigens were conserved in the core genome, and strains’ antigenic profiles were generally stable. No strong evidence was found for any epistasis between antigens driving population dynamics, or redundancy between functionally similar accessory ABTs, or age stratification of antigen profiles. These results highlight the paradox of why substantial variation is observed in only a subset of epitopes. This result may indicate only some interactions between immunoglobulins and ABTs clear pneumococcal colonization or that acquired immunity to pneumococci is an accumulation of individually weak responses to ABTs evolving under different levels of functional constraint.

Pneumococcal conjugate vaccines (PCVs) used in childhood vaccination programs have resulted in replacement of vaccine-type with nonvaccine-type pneumococci in carriage and invasive pneumococcal disease (IPD). A vaccine based on highly conserved and protective pneumococcal antigens is urgently needed. Here, we performed intranasal immunization of mice with pneumococcal membrane particles (MPs) to mimic natural nasopharyngeal immunization. MP immunization gave excellent serotype-independent protection against IPD that was antibody dependent but independent of the cytotoxin pneumolysin. Using Western blotting, immunoprecipitation, mass spectrometry, and different bacterial mutants, we identified the conserved lipoproteins MalX and PrsA as the main antigens responsible for cross-protection. Additionally, we found that omitting the variable surface protein and vaccine candidate PspA from MPs enhanced protective immune responses to the conserved proteins. Our findings suggest that MPs containing MalX and PrsA could serve as a platform for pneumococcal vaccine development targeting the elderly and immunocompromised.

To test vaccines, formulated with novel antigens, to protect mice against Chlamydia infections. To determine the ability of polymorphic membrane proteins (Pmps) to induce cross-species protective immune responses, recombinant fragments from all nine C. trachomatis serovar E Pmps were used to vaccinate BALB/c mice utilizing CpG-1826 and Montanide ISA 720 as adjuvants. C. muridarum recombinant MOMP and PBS, formulated with the same adjuvants, were used as positive and negative controls, respectively. Mice were challenged intranasally with 104 inclusion-forming units (IFU) of C. muridarum. Animals were weighed daily and at 10days post-challenge, they were euthanized, their lungs harvested, weighed and the number of chlamydial IFU counted. Following vaccination the nine Pmps elicited immune responses. Based on body weight changes, or number of IFU recovered from lungs, mice vaccinated with Pmp C, G or H were the best protected. For example, over the 10-day period, the negative control group vaccinated with PBS lost significantly more body weight than mice immunized with PmpC or G (P<0.05). C. muridarum MOMP vaccinated mice were better protected against body weight losses than any group immunized with Pmps. Also, the median number of IFU recovered from the lungs of mice vaccinated with PmpC (72×106) or PmpH (61×106) was significantly less than from mice immunized with PBS (620×106; P<0.05). As determined by the number of IFU, all Pmps elicited less protection than C. muridarum MOMP (0.078×106 IFU; P<0.05). This is the first time PmpC has been shown to elicit cross-species protection against a respiratory challenge. Additional work with Pmps C, G and H is recommended to determine their ability to protect animal models against genital and ocular challenges.

Summary Anaplasma phagocytophilum, which causes granulocytic anaplasmosis in humans and animals, is a tick‐transmitted obligate intracellular bacterium that mediates its own uptake into neutrophils and non‐phagocytic cells. Invasins of obligate intracellular pathogens are attractive targets for protecting against or curing infection because blocking the internalization step prevents survival of these organisms. The complement of A. phagocytophilum invasins is incompletely defined. Here, we report the significance of a novel A. phagocytophilum invasion protein, AipA. A. phagocytophilum induced aipA expression during transmission feeding of infected ticks on mice. The bacterium upregulated aipA transcription when it transitioned from its non‐infectious reticulate cell morphotype to its infectious dense‐cored morphotype during infection of HL‐60 cells. AipA localized to the bacterial surface and was expressed during in vivo infection. Of the AipA regions predicted to be surface‐exposed, only residues 1 to 87 (AipA1–87) were found to be essential for host cell invasion. Recombinant AipA1–87 protein bound to and competitively inhibited A. phagocytophilum infection of mammalian cells. Antiserum specific for AipA1–87, but not other AipA regions, antagonized infection. Additional blocking experiments using peptide‐specific antisera narrowed down the AipA invasion domain to residues 9 to 21. An antisera combination targeting AipA1–87 together with two other A. phagocytophilum invasins, OmpA and Asp14, nearly abolished infection of host cells. This study identifies AipA as an A. phagocytophilum surface protein that is critical for infection, demarcates its invasion domain, and establishes a rationale for targeting multiple invasins to protect against granulocytic anaplasmosis. Anaplasma phagocytophilum is an obligate intracellular bacterium that utilizes multiple surface proteins called invasins that act cooperatively to mediate entry into host cells. Blocking infection therefore requires simultaneous targeting of the relevant invasins. Herein, we identify a novel A. phagocytophilum invasin, AipA, delineate its invasion domain, and demonstrate that antibodies targeting AipA together with two additional invasins – OmpA and Asp14 – block infection of host cells. Our work evidences the potential of multi‐target approaches for preventing infections by intracellular bacteria.

•An attenuated Brucella melitensis vaccine M5-90 has been mostly used for animals in China.•Immune response to BP26 and OMP31 of the vaccine was evaluated in sheep.•Specific antibody and IFN-γ response were detected at low levels in vaccinated sheep.•Protective efficacy of the vaccine was overall weak and shortlived.•These data are helpful to design a safer and more effective B. melitensis vaccine. In recent years, the number of cases of human brucellosis has been increasing by approximately 10% per year in China. Most cases were caused by Brucella melitensis through contacts with infected sheep, goats or their products. An attenuated B. melitensis vaccine M5-90 is currently used to vaccinate both animals in China. This vaccine has not been investigated for critical parameters such as immune response and its association with protective efficacy. In this study, humoral and cellular immune response to the periplasmic protein BP26 and the outer membrane protein OMP31 were evaluated in M5-90 vaccinated Chinese merino and Kazak sheep. Antibodies to BP26 or OMP31 were detected at low levels, and specific IFN-γ response was quantified. Strongly reactive peptides derived from BP26 and OMP31 identified five T-cell epitopes (BP26-6, -8, -11, -12 and OMP31-23) common to both sheep species, five species-specific epitopes (BP26-10, -18, -21 and -22 and OMP31-12) and four animal-specific epitopes (BP26-15, -23, OMP31-6 and -21), which stimulated specific IFN-γ response in vaccinated sheep. Among those T-cell epitopes, reactivity to BP26-18 and -21 epitopes was significantly associated with MHC-I B allele (P=0.024). However, a specific T-cell response induced by the M5-90 vaccine was relatively week and did not sustain long enough, which might be suppressed by rapid activation of T-regulatory (Treg) cells following vaccination. These findings provide an insight in designing a safer and more effective vaccine for use in animals and in humans.

Sepsis, severe sepsis and septic shock are the main cause of mortality in non-cardiac intensive care units. Immunometabolism has been linked to sepsis; however, the precise mechanism by which metabolic reprogramming regulates the inflammatory response is unclear. Here we show that aerobic glycolysis contributes to sepsis by modulating inflammasome activation in macrophages. PKM2-mediated glycolysis promotes inflammasome activation by modulating EIF2AK2 phosphorylation in macrophages. Pharmacological and genetic inhibition of PKM2 or EIF2AK2 attenuates NLRP3 and AIM2 inflammasomes activation, and consequently suppresses the release of IL-1β, IL-18 and HMGB1 by macrophages. Pharmacological inhibition of the PKM2–EIF2AK2 pathway protects mice from lethal endotoxemia and polymicrobial sepsis. Moreover, conditional knockout of PKM2 in myeloid cells protects mice from septic death induced by NLRP3 and AIM2 inflammasome activation. These findings define an important role of PKM2 in immunometabolism and guide future development of therapeutic strategies to treat sepsis. Inflammation involves a Warburg effect that switches cellular metabolism to glycolysis. Here the authors show this switch drives IL-1β, IL-18 and HMGB1 release from macrophages by activating the NLRP3 and AIM2 inflammasomes via protein kinase R phosphorylation, a pathway that can be inhibited to prevent sepsis in mice.

Iron availability plays an essential role in staphylococcal pathogenesis. We selected FhuD2, a lipoprotein involved in iron-hydroxamate uptake, as a novel vaccine candidate against Staphylococcus aureus. Unprecedented for staphylococcal lipoproteins, the protein was demonstrated to have a discrete, punctate localization on the bacterial surface. FhuD2 vaccination generated protective immunity against diverse clinical S. aureus isolates in murine infection models. Protection appeared to be associated with functional antibodies that were shown to mediate opsonophagocytosis, to be effective in passive transfer experiments, and to potentially block FhuD2-mediated siderophore uptake. Furthermore, the protein was found to be up-regulated in infected tissues and was required for staphylococcal dissemination and abscess formation. Herein we show that the staphylococcal iron-hydroxamate uptake system is important in invasive infection and functions as an efficacious vaccine target.