No abstract available

Dengue viruses (DENV) are the most important mosquito transmitted viral pathogens infecting humans. DENV infection produces a spectrum of disease, most commonly causing a self-limiting flu-like illness known as dengue fever; yet with increased frequency, manifesting as life-threatening dengue hemorrhagic fever (DHF). Waning cross-protective immunity from any of the four dengue serotypes may enhance subsequent infection with another heterologous serotype to increase the probability of DHF. Decades of effort to develop dengue vaccines are reaching the finishing line with multiple candidates in clinical trials. Nevertheless, concerns remain that imbalanced immunity, due to the prolonged prime-boost schedules currently used in clinical trials, could leave some vaccinees temporarily unprotected or with increased susceptibility to enhanced disease. Here we develop a DENV serotype 1 (DENV-1) DNA vaccine with the immunodominant cross-reactive B cell epitopes associated with immune enhancement removed. We compare wild-type (WT) with this cross-reactivity reduced (CRR) vaccine and demonstrate that both vaccines are equally protective against lethal homologous DENV-1 challenge. Under conditions mimicking natural exposure prior to acquiring protective immunity, WT vaccinated mice enhanced a normally sub-lethal heterologous DENV-2 infection resulting in DHF-like disease and 95% mortality in AG129 mice. However, CRR vaccinated mice exhibited redirected serotype-specific and protective immunity, and significantly reduced morbidity and mortality not differing from naїve mice. Thus, we demonstrate in an in vivo DENV disease model, that non-protective vaccine-induced immunity can prime vaccinees for enhanced DHF-like disease and that CRR DNA immunization significantly reduces this potential vaccine safety concern. The sculpting of immune memory by the modified vaccine and resulting redirection of humoral immunity provide insight into DENV vaccine-induced immune responses.

During infection with non-enveloped viruses, antibodies stimulate immunity from inside cells by activating the cytosolic Fc receptor TRIM21. This intracellular humoral response relies on opsonized viral particles reaching the cytosol intact but the antigenic and kinetic constraints involved are unknown. We have solved the structure of a potent TRIM21-dependent neutralizing antibody in complex with human adenovirus 5 hexon and show how these properties influence immune activity. Structure-guided mutagenesis was used to generate antibodies with 20,000-fold variation in affinity, on-rates that differ by ~50-fold and off-rates by >175-fold. Characterization of these variants during infection revealed that TRIM21-dependent neutralization and NFκB activation was largely unaffected by on-rate kinetics. In contrast, TRIM21 antiviral activity was exquisitely dependent upon off-rate, with sub-μM affinity antibodies nevertheless unable to stimulate signaling because of fast dissociation kinetics. These results define the antibody properties required to elicit an efficient intracellular immune response during viral infection.

Colorectal cancer (CRC) is the third most lethal cancer in the world, and its incidence is steadily rising. In this study, we investigated the induction of humoral immunity by a phytogalactolipid enriched fraction (CRA) derived from the medicinal plant Crassocephalum rabens (Benth.) S. Moore to combat CRC.

Transplantation is used to treat many different diseases; however, without the use of immunosuppressants, which can be toxic to the patient, grafted tissue is rejected by the immune system. Humoral immune responses, particularly antibodies and complement, are significant components in rejection. Remarkably, Sertoli cells (SCs), immunoregulatory testicular cells, survive long-term after transplantation without immunosuppression. The objective of this study was to assess SC regulation of these humoral-based immune factors. Mouse SCs survived in vitro human complement (model of robust complement-mediated rejection) and survived in vivo as allografts with little-to-no antibody or complement fragment deposition. Microarray data and ELISA analyses identified at least 14 complement inhibitory proteins expressed by mouse SCs, which inhibit complement at multiple points. Interestingly, a mouse SC line (MSC-1), which was rejected by day 20 post transplantation, also survived in vitro human complement, showed limited deposition of antibodies and complement, and expressed complement inhibitors. Together this suggests that SC inhibition of complement-mediated killing is an important component of SC immune regulation. However, other mechanisms of SC immune modulation are also likely involved in SC graft survival. Identifying the mechanisms that SCs use to achieve extended survival as allografts could be utilized to improve graft survival.

Autophagy is a process that promotes the lysosomal degradation of cytoplasmic proteins and is highly conserved in eukaryotic organisms. Autophagy maintains homeostasis in organisms and regulates multiple developmental processes, and autophagy disruption is related to human diseases. However, the functional roles of autophagy in mediating innate immune responses are largely unknown. In this study, we sought to understand how Atg2, an autophagy-related gene, functions in the innate immunity of Drosophila melanogaster. The results showed that a large number of melanotic nodules were produced upon inhibition of Atg2. In addition, inhibiting Atg2 suppressed the phagocytosis of latex beads, Staphylococcus aureus and Escherichia coli; the proportion of Nimrod C1 (one of the phagocytosis receptors)-positive hemocytes also decreased. Moreover, inhibiting Atg2 altered actin cytoskeleton patterns, showing longer filopodia but with decreased numbers of filopodia. The expression of AMP-encoding genes was altered by inhibiting Atg2. Drosomycin was upregulated, and the transcript levels of Attacin-A, Diptericin and Metchnikowin were decreased. Finally, the above alterations caused by the inhibition of Atg2 prevented flies from resisting invading pathogens, showing that flies with low expression of Atg2 were highly susceptible to Staphylococcus aureus and Erwinia carotovora carotovora 15 infections. In conclusion, Atg2 regulated both cellular and humoral innate immunity in Drosophila. We have identified Atg2 as a crucial regulator in mediating the homeostasis of immunity, which further established the interactions between autophagy and innate immunity.

Conventional models suggest that long-term antibody responses are maintained by the continuous differentiation of memory B cells into antibody-secreting plasma cells. This is based on the notion that plasma cells are short-lived and need to be continually replenished by memory B cells. We examined the issue of plasma cell longevity by following the persistence of LCMV-specific antibody and plasma cell numbers after in vivo depletion of memory B cells and by adoptive transfer of virus-specific plasma cells into naive mice. The results show that a substantial fraction of plasma cells can survive and continue to secrete antibody for extended periods of time (>1 year) in the absence of any detectable memory B cells. This study documents the existence of long-lived plasma cells and demonstrates a new mechanism by which humoral immunity is maintained.

Tumour cells transfected with cDNAs encoding non-self proteins were used to investigate the ability of the immune system to respond to immunogenic antigens expressed by tumours. Secreted, intracellular and surface proteins were used as model antigens, as these reflect the potential forms of tumour antigens. Syngeneic BALB/c mice injected with viable line 1 lung carcinoma or EMT6 mammary tumour cells secreting ovalbumin (OVA) or prostate-specific antigen (PSA) produced very high immunoglobulin G (IgG) antibody titres, equivalent to those of mice injected with protein in Freund's complete adjuvant (FCA). Secretion of the antigens was not necessary as tumour cells expressing a cell-surface antigen (HER-2/Neu) or an intracellular antigen - green fluorescence protein (GFP) - also generated high-titre antigen-specific IgG antibodies. In interleukin-4 (IL-4)-deficient mice, both IgG1 and IgG2a were produced in response to OVA administered in FCA, whereas in response to tumour-produced antigen, the antibodies switched from predominantly IgG1 to IgG2a, indicating that the mechanisms responsible for antibody induction differed between these forms of immunization. In contrast to the line 1 and EMT6 tumours, which are of BALB/c origin, OVA- or PSA-producing B16 melanoma cells, which are of C57BL/6 origin, failed to elicit antibody production. This was not the result of strain differences, as a similar finding was observed when the tumours were grown in (BALB/c x C57BL/6)F1 mice, but appeared to be caused by intrinsic differences in the tumours. Furthermore, co-injection of both B16/OVA and line 1 tumours resulted in production of anti-OVA antibody, indicating that B16 tumours were not immunosuppressive, but instead line 1 tumours appear to exert an adjuvant effect.

The proliferation of antigen-specific lymphocytes and resulting clonal expansion are essential for adaptive immunity. We report here that B cell-specific deletion of the heavy chain of CD98 (CD98hc) resulted in lower antibody responses due to total suppression of B cell proliferation and subsequent plasma cell formation. Deletion of CD98hc did not impair early B cell activation but did inhibit later activation of the mitogen-activated protein kinase Erk1/2 and downregulation of the cell cycle inhibitor p27. Reconstitution of CD98hc-deficient B cells with CD98hc mutants showed that the integrin-binding domain of CD98hc was required for B cell proliferation but that the amino acid-transport function of CD98hc was dispensable for this. Thus, CD98hc supports integrin-dependent rapid proliferation of B cells. We propose that the advantage of adaptive immunity favored the appearance of CD98hc in vertebrates.

Bacteriophages are promising antibacterial agents. Although they have been recognized as bacterial viruses and are considered to be non-interacting with eukaryotic cells, there is growing evidence that phages may have a significant impact on the immune system via interactions with macrophages, neutrophils, and T-cell polarization. In this study, the influence of phages of podovirus, siphovirus, and myovirus morphotypes on humoral immunity of CD-1 mice was investigated. In addition, tissue distribution of the phages was tested in these mice. No common patterns were found either in the distribution of phages in mice or in changes in the levels of cytokines in the sera of mice once injected with phages. Importantly, pre-existing IgM-class antibodies directed against capsid proteins of phages with myovirus and siphovirus morphotypes were identified in mice before immunization. After triple immunization of CD1-mice with phages without any adjuvant, levels of anti-phage serum polyclonal IgG antibodies increased. Immunogenic phage proteins recognized by IgM and/or IgG antibodies were identified using Western blot analysis and mass spectrometry. In addition, mice serum collected after immunization demonstrated neutralizing properties, leading to a substantial decrease in infectivity of investigated phages with myovirus and siphovirus morphotypes. Moreover, serum samples collected before administration of these phages exhibited some ability to reduce the phage infectivity. Furthermore, Proteus phage PM16 with podovirus morphotype did not elicit IgM or IgG antibodies in immunized mice, and no neutralizing activities against PM16 were revealed in mouse serum samples before and after immunization.

Foxp3+ regulatory T cells (Tregs) are crucial for maintaining tolerance to self-antigens and preventing autoimmune diseases. Loss of Foxp3 expression leads to autoimmunity and disrupts humoral immune responses, including hyperproduction of immunoglobulin E (IgE). Elucidation of how Tregs control antibody production can lead to the development of new therapies for autoimmune and allergic diseases. However, premature death of Foxp3-deficient mice makes it difficult to analyze the roles of Tregs in humoral immunity of adult mice. In this study, we developed Foxp3 conditional-knockout mice (Foxp3floxR26CreERT2) in which the Foxp3 gene was inducibly deleted by tamoxifen administration. After oral administration of tamoxifen, titers of immunoglobulins, particularly IgG2c and IgE, were increased in Foxp3floxR26CreERT2 mice compared with that in controls. Under these conditions, CD4+ T cells from Foxp3floxR26CreERT2 mice had increased expression of several activation markers, including inducible costimulator and CD40 ligand, as well as the cytokines interleukin 4 and interferon gamma. In addition, the proportions of T follicular helper (Tfh) cells and germinal center (GC) B cells were increased in Foxp3floxR26CreERT2 mice compared with those in controls. These results indicated that Tregs controlled excessive or pathogenic antibody production by suppressing Tfh cell differentiation and GC formation. Furthermore, these data suggested that Foxp3floxR26CreERT2 mice could be a useful tool for screening therapeutic agents.

Adjuvants are central to the efficacy of subunit vaccines. Aluminum hydroxide (alum) is the most commonly used vaccine adjuvant, yet its adjuvanticity is often weak and mechanisms of triggering antibody responses remain poorly understood. We demonstrate that site-specific modification of immunogens with short peptides composed of repeating phosphoserine (pSer) residues enhances binding to alum and prolongs immunogen bioavailability. The pSer-modified immunogens formulated in alum elicited greatly increased germinal center, antibody, neutralizing antibody, memory and long-lived plasma cell responses compared to conventional alum-adsorbed immunogens. Mechanistically, pSer-immunogen:alum complexes form nanoparticles that traffic to lymph nodes and trigger B cell activation through multivalent and oriented antigen display. Direct uptake of antigen-decorated alum particles by B cells upregulated antigen processing and presentation pathways, further enhancing B cell activation. These data provide insights into mechanisms of action of alum and introduce a readily translatable approach to significantly improve humoral immunity to subunit vaccines using a clinical adjuvant.

Long-term humoral immunity is mediated by short-lived plasma cells (replenished by memory B cells) and long-lived plasma cells. Their relative contributions are uncertain for immunity to SARS-CoV-2, especially given the widespread use of novel mRNA vaccines. Yet, this has far-reaching implications in terms of the need for regular booster doses in the general population and perhaps even revaccination in patients receiving B cell-depleting therapy. We aimed to characterise anti-SARS-CoV-2 antibody titres in patients receiving Rituximab following previous SARS-CoV-2 vaccination. We recruited 10 fully vaccinated patients (age: 16.9 ± 2.52 years) with childhood-onset nephrotic syndrome, not in relapse, receiving Rituximab for their steroid/calcineurin-inhibitor sparing effect. Antibodies to SARS-CoV-2 spike (S) and nucleocapsid (N) proteins were measured immediately prior to Rituximab and again ~6 months later, using the Roche Elecys® Anti-SARS-CoV-2 (S) assay. All ten patients were positive for anti-S antibodies prior to Rituximab, with six patients (60%) having titres above the upper limit of detection (>12,500 U/mL). Following Rituximab therapy, there was a reduction in anti-S titres (p = 0.043), but all patients remained positive for anti-S antibodies, with five patients (50%) continuing to have titres >12,500 U/mL. Six patients (60%) were positive for anti-N antibodies prior to Rituximab. Following Rituximab therapy, only three of these six patients remained positive for anti-N antibodies (p = 0.036 compared to anti-S seroreversion). Humoral immunity to SARS-CoV-2 is likely to be mediated in part by long-lived plasma cells.

AAV vectors have shown great promise for clinical gene therapy (GT), but pre-existing human immunity against the AAV capsid often limits transduction. Thus, testing promising AAV-based GT approaches in an animal model with similar pre-existing immunity could better predict clinical outcome. Sheep have long been used for basic biological and preclinical studies. Moreover, we have re-established a line of sheep with severe hemophilia A (HA). Given the impetus to use AAV-based GT to treat hemophilia, we characterized the pre-existing ovine humoral immunity to AAV. ELISA revealed naturally-occurring antibodies to AAV1, AAV2, AAV5, AAV6, AAV8, and AAV9. For AAV2, AAV8, and AAV9 these inhibit transduction in a luciferase-based neutralization assay. Epitope mapping identified peptides that were common to the capsids of all AAV serotypes tested (AAV2, AAV5, AAV8 and AAV9), with each animal harboring antibodies to unique and common capsid epitopes. Mapping using X-ray crystallographic AAV capsid structures demonstrated that these antibodies recognized both surface epitopes and epitopes located within regions of the capsid that are internal or buried in the capsid structure. These results suggest that sheep harbor endogenous AAV, which induces immunity to both intact capsid and to capsid epitopes presented following proteolysis during the course of infection. In conclusion, their clinically relevant physiology and the presence of naturally-occurring antibodies to multiple AAV serotypes collectively make sheep a unique model in which to study GT for HA, and other diseases, and develop strategies to circumvent the clinically important barrier of pre-existing AAV immunity.

Norovirus (NoV) P domain complexes, the 24 mer P particles and the P dimers, induced effective humoral immunity, but their role in the cellular immune responses remained unclear. We reported here a study on cellular immune responses of the two P domain complexes in comparison with the virus-like particle (VLP) of a GII.4 NoV (VA387) in mice. The P domain complexes induced significant central memory CD4(+) T cell phenotypes (CD4(+) CD44(+) CD62L(+) CCR7(+)) and activated polyclonal CD4(+) T cells as shown by production of Interleukin (IL)-2, Interferon (IFN)-γ, and Tumor Necrosis Factor (TNF)-α. Most importantly, VA387-specific CD4(+) T cell epitope induced a production of IFN-γ, indicating an antigen-specific CD4(+) T cell response in P domain complex-immunized mice. Furthermore, P domain complexes efficiently induced bone marrow-derived dendritic cell (BMDC) maturation, evidenced by up-regulation of co-stimulatory and MHC class II molecules, as well as production of IL-12 and IL-1β. Finally, P domain complex-induced mature dendritic cells (DCs) elicited proliferation of specific CD4(+) T cells targeting VA387 P domain. Overall, we conclude that the NoV P domain complexes are efficiently presented by DCs to elicit not only humoral but also cellular immune responses against NoVs. Since the P particle is highly effective for both humoral and cellular immune responses and easily produced in Escherichia coli (E. coli), it is a good choice of vaccine against NoVs and a vaccine platform against other diseases.

As important players in the host defense system, commensal microbes and the microbiota influence multiple aspects of host physiology. Bordetella pertussis infection is highly contagious among humans. However, the roles of the microbiota in B. pertussis pathogenesis are poorly understood. Here, we show that antibiotic-mediated depletion of the microbiota results in increased susceptibility to B. pertussis infection during the early stage. The increased susceptibility was associated with a marked impairment of the systemic IgG, IgG2a, and IgG1 antibody responses to B. pertussis infection after antibiotic treatment. Furthermore, the microbiota impacted the short-lived plasma cell responses as well as the recall responses of memory B cells to B. pertussis infection. Finally, we found that the dysbiosis caused by antibiotic treatment affects CD4+ T cell generation and PD-1 expression on CD4+ T cells and thereby perturbs plasma cell differentiation. Our results have revealed the importance of commensal microbes in modulating host immune responses to B. pertussis infection and support the possibility of controlling the severity of B. pertussis infection in humans by manipulating the microbiota.

Airway epithelial cells (AECs) display remarkable plasticity in response to infectious stimuli and their functional adaptations are critical for antimicrobial immunity. However, the roles of AECs and humoral mediators to host defense in non-communicable lung inflammation remain elusive. We dissected pulmonary defense against Streptococcus pneumoniae in hosts with pre-existing inflammatory conditions (SPC-HAxTCR-HA mice). Lung tissue transcriptomics and bronchoalveolar lavage fluid (BALF) proteomics revealed an induction of humoral defense mechanisms in inflamed lungs. Accordingly, besides antibacterial proteins and complement components being overrepresented in inflamed lungs, elevated polymeric immunoglobulin receptor (pIgR)-expression in AECs correlated with increased secretory immunoglobulin (SIg) transport. Consequently, opsonization assays revealed augmented pneumococcal coverage by SIgs present in the BALF of SPC-HAxTCR-HA mice, which was associated with enhanced antipneumococcal resistance. These findings emphasize the immunologic potential of AECs as well as their central role in providing antibacterial protection and put forward pIgR as potential target for therapeutic manipulation in infection-prone individuals.

By the beginning of 2021, the battle against coronavirus disease 2019 (COVID-19) remains ongoing. Investigating the adaptive immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, in patients who have recovered from this disease could contribute to our understanding of the natural host immune response. We enrolled 38 participants in this study. 7 healthy participants and 31 COVID-19 patients who had recovered from COVID-19 and categorized them into 3 groups according to their previous clinical presentations: 10 moderate, 9 mild, and 12 asymptomatic. Flow cytometry analysis of peripheral lymphocyte counts in recovered patients showed significantly increased levels of CD4+ T cells in patients with a history of mild and moderate COVID-19 symptoms compared with those healthy individuals (p < 0.05 and p < 0.0001 respectively). whereas no significant difference was observed in the CD8+ T cell percentage in COVID-19-recovered patients compared with healthy individuals. Our study demonstrated that antibodies against the SARS-CoV-2 spike protein (anti-S) IgG antibody production could be observed in all recovered COVID-19 patients, regardless of whether they were asymptomatic (p < 0.05)or presented with mild (p < 0.0001) or moderate symptoms (p < 0.01). Anti-S IgG antibodies could be detected in participants up to 90 days post-infection. In conclusion, the lymphocyte levels in recovered patients were associated with the clinical presentation of the disease, and further analysis is required to investigate relationships between different clinical presentations and lymphocyte activation and function.

To explore coevolution between the gut microbiota and the humoral immune system of the host, we used chickens as the model organism. The host populations were two lines (HAS and LAS) developed from a common founder that had undergone 40 generations of divergent selection for antibody titers to sheep red blood cells (SRBC) and two relaxed sublines (HAR and LAR). Analysis revealed that microevolution of host humoral immunity contributed to the composition of gut microbiota at the taxa level. Relaxing selection enriched some microorganisms whose functions were opposite to host immunity. Particularly, Ruminococcaceae and Oscillospira enriched in high antibody relaxed (HAR) and contributed to reduction in antibody response, while Lactobacillus increased in low antibody relaxed (LAR) and elevated the antibody response. Microbial functional analysis showed that alterations were involved in pathways relating to the immune system and infectious diseases. Our findings demonstrated co-microevolution relationships of host-microbiota and that gut microorganisms influenced host immunity.

Lyme Disease caused by infection with Borrelia burgdorferi is an emerging infectious disease and already by far the most common vector-borne disease in the U.S. Similar to many other infections, infection with B. burgdorferi results in strong antibody response induction, which can be used clinically as a diagnostic measure of prior exposure. However, clinical studies have shown a sometimes-precipitous decline of such antibodies shortly following antibiotic treatment, revealing a potential deficit in the host's ability to induce and/or maintain long-term protective antibodies. This is further supported by reports of frequent repeat infections with B. burgdorferi in endemic areas. The mechanisms underlying such a lack of long-term humoral immunity, however, remain unknown. We show here that B. burgdorferi infected mice show a similar rapid disappearance of Borrelia-specific antibodies after infection and subsequent antibiotic treatment. This failure was associated with development of only short-lived germinal centers, micro-anatomical locations from which long-lived immunity originates. These showed structural abnormalities and failed to induce memory B cells and long-lived plasma cells for months after the infection, rendering the mice susceptible to reinfection with the same strain of B. burgdorferi. The inability to induce long-lived immune responses was not due to the particular nature of the immunogenic antigens of B. burgdorferi, as antibodies to both T-dependent and T-independent Borrelia antigens lacked longevity and B cell memory induction. Furthermore, influenza immunization administered at the time of Borrelia infection also failed to induce robust antibody responses, dramatically reducing the protective antiviral capacity of the humoral response. Collectively, these studies show that B. burgdorferi-infection results in targeted and temporary immunosuppression of the host and bring new insight into the mechanisms underlying the failure to develop long-term immunity to this emerging disease threat.