Functional and transcriptional heterogeneity within the massively expanding HLADR+CD38+ CD8 T cell population in acute febrile dengue patients.

IMPORTANCE: CD8 T cells play a crucial role in protecting against intracellular pathogens such as viruses by eliminating infected cells and releasing anti-viral cytokines such as interferon gamma (IFNγ). Consequently, there is significant interest in comprehensively characterizing CD8 T cell responses in acute dengue febrile patients. Previous studies, including our own, have demonstrated that a discrete population of CD8 T cells with HLADR+ CD38+ phenotype undergoes massive expansion during the acute febrile phase of natural dengue virus infection. Although about a third of these massively expanding HLADR+ CD38+ CD8 T cells were also CD69high when examined ex vivo, only a small fraction of them produced IFNγ upon in vitro peptide stimulation. Therefore, to better understand such functional diversity of CD8 T cells responding to dengue virus infection, it is important to know the cytokines/chemokines expressed by these peptide-stimulated HLADR+CD38+ CD8 T cells and the transcriptional profiles that distinguish the CD69+IFNγ+, CD69+IFNγ-, and CD69-IFNγ- subsets.

Immunophenotyping and Transcriptional Profiling of Human Plasmablasts in Dengue.

Plasmablasts represent a specialized class of antibody-secreting effector B cells that transiently appear in blood circulation following infection or vaccination. The expansion of these cells generally tends to be massive in patients with systemic infections such as dengue or Ebola that cause hemorrhagic fever. To gain a detailed understanding of human plasmablast responses beyond antibody expression, here, we performed immunophenotyping and RNA sequencing (RNA-seq) analysis of the plasmablasts from dengue febrile children in India. We found that plasmablasts expressed several adhesion molecules and chemokines or chemokine receptors that are involved in endothelial interactions or homing to inflamed tissues, including skin, mucosa, and intestine, and upregulated the expression of several cytokine genes that are involved in leukocyte extravasation and angiogenesis. These plasmablasts also upregulated the expression of receptors for several B-cell prosurvival cytokines that are known to be induced robustly in systemic viral infections such as dengue, some of which generally tend to be relatively higher in patients manifesting hemorrhage and/or shock than in patients with mild febrile infection. These findings improve our understanding of human plasmablast responses during the acute febrile phase of systemic dengue infection. IMPORTANCE Dengue is globally spreading, with over 100 million clinical cases annually, with symptoms ranging from mild self-limiting febrile illness to more severe and sometimes life-threatening dengue hemorrhagic fever or shock, especially among children. The pathophysiology of dengue is complex and remains poorly understood despite many advances indicating a key role for antibody-dependent enhancement of infection. While serum antibodies have been extensively studied, the characteristics of the early cellular factories responsible for antibody production, i.e., plasmablasts, are only beginning to emerge. This study provides a comprehensive understanding of the transcriptional profiles of human plasmablasts from dengue patients.

Chikungunya-specific IgG and neutralizing antibody responses in natural infection of Chikungunya virus in children from India.

Chikungunya virus (CHIKV) infection is endemic in many different countries. CHIKV outbreaks are emerging in new areas and re-emerging in previously exposed geographical regions, thus making it a significant public health concern. CHIKV infections are often clinically inapparent, especially in children, which poses a challenge to testing and evaluating any vaccine. During CHIKV infection, CHIKV-specific antibodies are produced, and some of these antibodies can neutralize viruses released from infected cells before they can enter uninfected cells. In this study, we evaluated IgG binding and neutralizing antibody responses in paired serum samples from CHIKV-infected children and those with other febrile illness, using a recombinant truncated E2 protein and whole CHIKV particles as test antigens. Antibody detection using the truncated E2 protein showed a significant overlap between CHIKV-infected subjects and those with other febrile illnesses. This overlap was greater when binding antibody titers were determined using fixed CHIKV particles as the test antigen. Acute- and convalescent-phase sera collected from children after CHIKV infection showed significant differences in their neutralizing capacity. The neutralizing and binding antibody response showed a significant positive correlation. We detected IgG antibodies in most cases during the acute phase of infection. This was observed at two different geographical locations, one of which is not considered highly endemic. Conventional wisdom would suggest this to be a marker of re-infection (secondary infection). However, dissenting opinions have been voiced in other viral diseases (such as Ebola) where studies have detected IgG in acute illness. In the absence of any significant body of work documenting secondary CHIKV infections, we believe further work is needed to understand the early IgG response that we observed.

Epidemiology and molecular characterization of chikungunya virus from human cases in North India, 2016.

Chikungunya virus (CHIKV), an arthropod-borne Alphavirus is responsible for chikungunya disease. Arthralgia and arthritis are the major symptom. Some patients recover early while others for a very long time. This study provides, epidemiology and molecular characterization of three whole-genome sequences of CHIKV and assessed phylogenetic analysis, physiological properties, antigenicity, and B-cell epitope prediction by in silico. We report the clinical epidemiology of 325 suspected patients. Of these, 118 (36.30%) were confirmed CHIKV positive by either PCR or ELISA. Clinical analysis showed joint pain, joint swelling and headache were frequent and significant features. Phylogenie analysis showed the currently circulating strain is in close clustring to Africa, Uganda, and Singapore CHIKV strains. Molecular characterization by WGS was done. Thirty eight amino acid changes in the nonstructural proteins were found with respect to the S27 (ECSA) strain. Of these five located in nsP2. Similarly, 34 amino acid changes in structural proteins were observed. The major change was notice; in E3 protein hydropathicity -0.281 to -0.362, in E2 isoelectric point (pI) 8.24 to 8.37, instability index 66.08 to 71.062, aliphatic index varied from 74.69 to 68.59 and E3 75.79 to 70.05. In nsP1 protein pI varies from 6.62 to 8.04, while no other change was observed in structural and nonstructural protein. The linear B-cell epitopes, position, and number varied with the mutation. The molecular characterizations of WGS demonstrate the observation of protein, antigenicity with respect to the mutation.

Human Cytomegalovirus Enhances Placental Susceptibility and Replication of Human Immunodeficiency Virus Type 1 (HIV-1), Which May Facilitate In Utero HIV-1 Transmission.

Several co-pathogens that pose threats to the fetus during gestation, including human cytomegalovirus (HCMV), may also contribute to mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1). Within endemic settings, associations between maternal HCMV viral load and increased incidence of MTCT of HIV-1 are documented; however, the mechanisms that promote transmission are poorly characterized. Here we demonstrate that HCMV coinfection enhances susceptibility and viral replication of HIV-1 in placental macrophages (Hofbauer cells) in vitro. Consistent with enhanced viral susceptibility, HCMV exposure upregulates CCR5 and CD80 expression on Hofbauer cells. HCMV also significantly induces type I interferon (IFN), proinflammatory cytokines, and antiviral gene expression. Interestingly, we found that HCMV diminishes type I IFN-mediated phosphorylation of STAT2. Collectively, our data suggest that HCMV-induced activation, local inflammation, and antagonism of type I IFN responses in placental Hofbauer cells promote in utero transmission of HIV-1.

Immune Priming and Long-term Persistence of Memory B Cells After Inactivated Poliovirus Vaccine in Macaque Models: Support for at least 2 Doses.

Background: As a risk-mitigation strategy to minimize paralytic polio following withdrawal of Sabin type 2 from the oral poliovirus vaccine in April 2016, a single full dose or 2 fractional doses of inactivated poliovirus vaccine (IPV) are recommended. However, limited knowledge exists on long-term persistence of immune memory following 1- or 2-dose IPV schedules. Methods: We examined induction and maintenance of immune memory following single- vs 2-dose IPV schedules, either full-dose intramuscular or fractional-dose intradermal, in rhesus macaques. Humoral responses, bone marrow-homing antibody-secreting plasma cells, and blood-circulating/lymph node-homing memory B cells were examined longitudinally. Results: A single dose of IPV, either full or fractional, induced binding antibodies and memory B cells in all vaccinated macaques, despite failing to induce neutralizing antibodies (NT Abs) in many of them. However, these memory B cells declined rapidly, reaching below detection in the systemic circulation by 5 months; although a low frequency of memory B cells was detectable in draining lymph nodes of some, but not all, animals. By contrast, a 2-dose vaccination schedule, either full or fractional, efficiently induced NT Abs in all animals along with bone marrow-homing plasma cells and memory B cells. These memory B cells persisted in the systemic circulation for up to 16 months, the maximum duration tested after the second dose of vaccination. Conclusions: Two doses of IPV, regardless of whether fractional or full, are more effective than a single dose for inducing long-lasting memory B cells.

Virus binding to a plasma membrane receptor triggers interleukin-1 alpha-mediated proinflammatory macrophage response in vivo.

The recognition of viral components by host pattern-recognition receptors triggers the induction of the antiviral innate immune response. Toll-like receptor 9 (TLR9) and NLRP3 inflammasome were shown to be the principal specific sensors of viral double-stranded DNA. Here we present evidence that macrophages in vivo activated an innate immune response to a double-stranded DNA virus, adenovirus (Ad), independently of TLR9 or NLRP3 inflammasome. In response to Ad, macrophage-derived IL-1 alpha triggered IL-1RI-dependent production of a defined set of proinflammatory cytokines and chemokines. The IL-1 alpha-mediated response required a selective interaction of virus arginine-glycine-aspartic acid (RGD) motifs with macrophage beta(3) integrins. Thus, these data identify IL-1 alpha-IL-1RI as a key pathway allowing for the activation of proinflammatory responses to the virus, independently of its genomic nucleic acid recognition.

Characterization of Human CD8 T Cell Responses in Dengue Virus-Infected Patients from India.

Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR- CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-γ) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-γ unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-γ by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-γ unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue. IMPORTANCE: Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-γ in vitro Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects.

CpG oligonucleotide-mediated co-stimulation of mouse invariant natural killer T cells negatively regulates their activation status.

Invariant natural killer T (iNKT) cells play important roles in antimicrobial defense and immune-regulation. We have previously shown that iNKT cells express certain toll-like receptors (TLR), and that TLR co-stimulation of iNKT cells in the presence of suboptimal concentrations of T cell receptor (TCR) agonists enhances cellular activation. In the present study, we investigated the regulatory effects of CpG oligonucleotides in mouse primary hepatic and splenic iNKT cells and in DN32.D3 iNKT cells. We show that CpG treatment of iNKT cells in the presence of higher concentrations of TCR agonists (α-GalCer or anti-CD3 mAb) results in the up-regulation of TLR9 in iNKT cells with a concurrent reduction in their cellular activation, as assessed by their production of IL-2, IL-4 and IFN-γ compared with controls. CpG-mediated down-regulation of iNKT cell activation has been found to depend, at least in part, on signaling by MyD88, a critical adapter moiety downstream of TLR9 signaling. Mechanistically, iNKT cells treated with CpG in the presence of TCR agonists show inhibition of MAPK signaling as determined by the levels of ERK1/2 and p38 MAPKs. Furthermore, CpG treatment leads to an increased induction of phosphatases, DUSP1 and SHP-1, that seem to impede MAPK and TCR signaling, resulting in the negative regulation of iNKT cell activation. Our findings therefore suggest a novel regulatory role for CpG in iNKT cells in the mediation of a negative feedback mechanism to control overactive iNKT cell responses and hence to avoid undesirable excessive immunopathology.

Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines.

To explore the human T cell response to acute viral infection, we performed a longitudinal analysis of CD8(+) T cells responding to the live yellow fever virus and smallpox vaccines--two highly successful human vaccines. Our results show that both vaccines generated a brisk primary effector CD8(+) T cell response of substantial magnitude that could be readily quantitated with a simple set of four phenotypic markers. Secondly, the vaccine-induced T cell response was highly specific with minimal bystander effects. Thirdly, virus-specific CD8(+) T cells passed through an obligate effector phase, contracted more than 90% and gradually differentiated into long-lived memory cells. Finally, these memory cells were highly functional and underwent a memory differentiation program distinct from that described for human CD8(+) T cells specific for persistent viruses. These results provide a benchmark for CD8(+) T cell responses induced by two of the most effective vaccines ever developed.

Blocking lymphocyte trafficking with FTY720 prevents inflammation-sensitized hypoxic-ischemic brain injury in newborns.

Intrauterine infection (chorioamnionitis) aggravates neonatal hypoxic-ischemic (HI) brain injury, but the mechanisms linking systemic inflammation to the CNS damage remain uncertain. Here we report evidence for brain influx of T-helper 17 (TH17)-like lymphocytes to coordinate neuroinflammatory responses in lipopolysaccharide (LPS)-sensitized HI injury in neonates. We found that both infants with histological chorioamnionitis and rat pups challenged by LPS/HI have elevated expression of the interleukin-23 (IL-23) receptor, a marker of early TH17 lymphocytes, in the peripheral blood mononuclear cells. Post-LPS/HI administration of FTY720 (fingolimod), a sphingosine-1-phosphate receptor agonist that blocks lymphocyte trafficking, mitigated the influx of leukocytes through the choroid plexus and acute induction of nuclear factor-κB signaling in the brain. Subsequently, the FTY720 treatment led to attenuated blood-brain barrier damage, fewer cluster of differentiation 4-positive, IL-17A-positive T-cells in the brain, less proinflammatory cytokine, and better preservation of growth and white matter functions. The FTY720 treatment also provided dose-dependent reduction of brain atrophy, rescuing >90% of LPS/HI-induced brain tissue loss. Interestingly, FTY720 neither opposed pure-HI brain injury nor directly inhibited microglia in both in vivo and in vitro models, highlighting its unique mechanism against inflammation-sensitized HI injury. Together, these results suggest that the dual hit of systemic inflammation and neonatal HI injury triggers early onset of the TH17/IL-17-mediated immunity, which causes severe brain destruction but responds remarkably to the therapeutic blockade of lymphocyte trafficking.

Activation of the RIG-I pathway during influenza vaccination enhances the germinal center reaction, promotes T follicular helper cell induction, and provides a dose-sparing effect and protective immunity.

UNLABELLED: Pattern recognition receptors (PRR) sense certain molecular patterns uniquely expressed by pathogens. Retinoic-acid-inducible gene I (RIG-I) is a cytosolic PRR that senses viral nucleic acids and induces innate immune activation and secretion of type I interferons (IFNs). Here, using influenza vaccine antigens, we investigated the consequences of activating the RIG-I pathway for antigen-specific adaptive immune responses. We found that mice immunized with influenza vaccine antigens coadministered with 5'ppp-double-stranded RNA (dsRNA), a RIG-I ligand, developed robust levels of hemagglutination-inhibiting antibodies, enhanced germinal center reaction, and T follicular helper cell responses. In addition, RIG-I activation enhanced antibody affinity maturation and plasma cell responses in the draining lymph nodes, spleen, and bone marrow and conferred protective immunity against virus challenge. Importantly, activation of the RIG-I pathway was able to reduce the antigen requirement by 10- to 100-fold in inducing optimal influenza-specific cellular and humoral responses, including protective immunity. The effects induced by 5'ppp-dsRNA were significantly dependent on type I IFN and IPS-1 (an adapter protein downstream of the RIG-I pathway) signaling but were independent of the MyD88- and TLR3-mediated pathways. Our results show that activation of the RIG-I-like receptor pathway programs the innate immunity to achieve qualitatively and quantitatively enhanced protective cellular adaptive immune responses even at low antigen doses, and this indicates the potential utility of RIG-I ligands as molecular adjuvants for viral vaccines. IMPORTANCE: The recently discovered RNA helicase family of RIG-I-like receptors (RLRs) is a critical component of host defense mechanisms responsible for detecting viruses and triggering innate antiviral cytokines that help control viral replication and dissemination. In this study, we show that the RLR pathway can be effectively exploited to enhance adaptive immunity and protective immune memory against viral infection. Our results show that activation of the RIG-I pathway along with influenza vaccination programs the innate immunity to induce qualitatively and quantitatively superior protective adaptive immunity against pandemic influenza viruses. More importantly, RIG-I activation at the time of vaccination allows induction of robust adaptive responses even at low vaccine antigen doses. These results highlight the potential utility of exploiting the RIG-I pathway to enhance viral-vaccine-specific immunity and have broader implications for designing better vaccines in general.

Just-in-time vaccines: Biomineralized calcium phosphate core-immunogen shell nanoparticles induce long-lasting CD8(+) T cell responses in mice.

Distributed and on-demand vaccine production could be game-changing for infectious disease treatment in the developing world by providing new therapeutic opportunities and breaking the refrigeration "cold chain". Here, we show that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize a biocompatible adjuvant in a single step. The resulting 50 nm calcium phosphate core-immunogen shell particles are comparable to soluble protein in inducing ovalbumin-specific antibody response and class switch recombination in mice. However, single dose vaccination with nanoparticles leads to higher expansion of ovalbumin-specific CD8(+) T cells upon challenge with an influenza virus bearing the ovalbumin-derived SIINFEKL peptide, and these cells produce high levels of IFN-γ. Furthermore, mice exhibit a robust antigen-specific CD8(+) T cell recall response when challenged with virus 8 months post-immunization. These results underscore the promise of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines. FROM THE CLINICAL EDITOR: This paper reports that a fusion protein between a calcium phosphate binding domain and the model antigen ovalbumin can mineralize into a biocompatible adjuvant in a single step, enabling distributed and on-demand vaccine production and eliminating the need for refrigeration of vaccines. The findings highlight the possibility of immunogen-controlled adjuvant mineralization for just-in-time manufacturing of effective T cell vaccines.

Regional Variation of the CD4 and CD8 T Cell Epitopes Conserved in Circulating Dengue Viruses and Shared with Potential Vaccine Candidates.

As dengue expands globally and many vaccines are under trials, there is a growing recognition of the need for assessing T cell immunity in addition to assessing the functions of neutralizing antibodies during these endeavors. While several dengue-specific experimentally validated T cell epitopes are known, less is understood about which of these epitopes are conserved among circulating dengue viruses and also shared by potential vaccine candidates. As India emerges as the epicenter of the dengue disease burden and vaccine trials commence in this region, we have here aligned known dengue specific T cell epitopes, reported from other parts of the world with published polyprotein sequences of 107 dengue virus isolates available from India. Of the 1305 CD4 and 584 CD8 epitopes, we found that 24% and 41%, respectively, were conserved universally, whereas 27% and 13% were absent in any viral isolates. With these data, we catalogued epitopes conserved in circulating dengue viruses from India and matched them with each of the six vaccine candidates under consideration (TV003, TDEN, DPIV, CYD-TDV, DENVax and TVDV). Similar analyses with viruses from Thailand, Brazil and Mexico revealed regional overlaps and variations in these patterns. Thus, our study provides detailed and nuanced insights into regional variation that should be considered for itemization of T cell responses during dengue natural infection and vaccine design, testing and evaluation.

Severe disease during both primary and secondary dengue virus infections in pediatric populations.

Dengue is a global epidemic causing over 100 million cases annually. The clinical symptoms range from mild fever to severe hemorrhage and shock, including some fatalities. The current paradigm is that these severe dengue cases occur mostly during secondary infections due to antibody-dependent enhancement after infection with a different dengue virus serotype. India has the highest dengue burden worldwide, but little is known about disease severity and its association with primary and secondary dengue infections. To address this issue, we examined 619 children with febrile dengue-confirmed infection from three hospitals in different regions of India. We classified primary and secondary infections based on IgM:IgG ratios using a dengue-specific enzyme-linked immunosorbent assay according to the World Health Organization guidelines. We found that primary dengue infections accounted for more than half of total clinical cases (344 of 619), severe dengue cases (112 of 202) and fatalities (5 of 7). Consistent with the classification based on binding antibody data, dengue neutralizing antibody titers were also significantly lower in primary infections compared to secondary infections (P ≤ 0.0001). Our findings question the currently widely held belief that severe dengue is associated predominantly with secondary infections and emphasizes the importance of developing vaccines or treatments to protect dengue-naive populations.

Light chain of a public SARS-CoV-2 class-3 antibody modulates neutralization against Omicron.

The pairing of antibody genes IGHV2-5/IGLV2-14 is established as a public immune response that potently cross-neutralizes SARS-CoV-2 variants, including Omicron, by targeting class-3/RBD-5 epitopes in the receptor binding domain (RBD). LY-CoV1404 (bebtelovimab) exemplifies this, displaying exceptional potency against Omicron sub-variants up to BA.5. Here, we report a human antibody, 002-S21B10, encoded by the public clonotype IGHV2-5/IGLV2-14. While 002-S21B10 neutralized key SARS-CoV-2 variants, it did not neutralize Omicron, despite sharing >92% sequence similarity with LY-CoV1404. The structure of 002-S21B10 in complex with spike trimer plus structural and sequence comparisons with LY-CoV1404 and other IGHV2-5/IGLV2-14 antibodies revealed significant variations in light-chain orientation, paratope residues, and epitope-paratope interactions that enable some antibodies to neutralize Omicron but not others. Confirming this, replacing the light chain of 002-S21B10 with the light chain of LY-CoV1404 restored 002-S21B10's binding to Omicron. Understanding such Omicron evasion from public response is vital for guiding therapeutics and vaccine design.

Molecular basis of SARS-CoV-2 Omicron variant evasion from shared neutralizing antibody response.

Understanding the molecular features of neutralizing epitopes is important for developing vaccines/therapeutics against emerging SARS-CoV-2 variants. We describe three monoclonal antibodies (mAbs) generated from COVID-19 recovered individuals during the first wave of the pandemic in India. These mAbs had publicly shared near germline gene usage and potently neutralized Alpha and Delta, poorly neutralized Beta, and failed to neutralize Omicron BA.1 SARS-CoV-2 variants. Structural analysis of these mAbs in complex with trimeric spike protein showed that all three mAbs bivalently bind spike with two mAbs targeting class 1 and one targeting a class 4 receptor binding domain epitope. The immunogenetic makeup, structure, and function of these mAbs revealed specific molecular interactions associated with the potent multi-variant binding/neutralization efficacy. This knowledge shows how mutational combinations can affect the binding or neutralization of an antibody, which in turn relates to the efficacy of immune responses to emerging SARS-CoV-2 escape variants.

Biomineralization and size control of stable calcium phosphate core-protein shell nanoparticles: potential for vaccine applications.

Calcium phosphate (CaP) polymorphs are nontoxic, biocompatible and hold promise in applications ranging from hard tissue regeneration to drug delivery and vaccine design. Yet, simple and robust routes for the synthesis of protein-coated CaP nanoparticles in the sub-100 nm size range remain elusive. Here, we used cell surface display to identify disulfide-constrained CaP binding peptides that, when inserted within the active site loop of Escherichia coli thioredoxin 1 (TrxA), readily and reproducibly drive the production of nanoparticles that are 50-70 nm in hydrodynamic diameter and consist of an approximately 25 nm amorphous calcium phosphate (ACP) core stabilized by the protein shell. Like bone and enamel proteins implicated in biological apatite formation, peptides supporting nanoparticle production were acidic. They also required presentation in a loop for high-affinity ACP binding as elimination of the disulfide bridge caused a nearly 3-fold increase in hydrodynamic diameters. When compared to a commercial aluminum phosphate adjuvant, the small core-shell assemblies led to a 3-fold increase in mice anti-TrxA titers 3 weeks postinjection, suggesting that they might be useful vehicles for adjuvanted antigen delivery to dendritic cells.

Homeostatic turnover of virus-specific memory CD8 T cells occurs stochastically and is independent of CD4 T cell help.

Memory CD8 T cells persist by Ag-independent homeostatic proliferation. To examine the dynamics of this cell turnover, we transferred lymphocytic choriomeningitis virus specific memory CD8 T cells into naive mice and analyzed their in vivo division kinetics longitudinally in individual recipients.Using mathematical modeling, we determined that proliferation of this stably maintained memory CD8 T cell population was homogeneous and stochastic with a small fraction of cells completing division at any given time with an intermitotic interval of 50 d. This homeostatic turnover was comparable between memory CD8 T cells of different viral epitope specificities and also the total memory phenotype (CD44(high)) CD8 T cells. It is well established that CD4 T cell help is critical for maintenance of CD8 T cells during chronic infections, but recent studies have suggested that CD4 T cell help is also required for maintenance of memory CD8 T cells following acute infections. Hence, we assessed the role of CD4 T cells in Ag-independent maintenance of memory CD8 T cells. Consistent with previous reports, we found that memory CD8 T cells declined when transferred into MHC class II-deficient mice. However, their numbers were maintained stably when transferred into CD4 T cell-deficient mice. Interestingly, their homeostatic proliferation, ability to make recall responses, and phenotype were independent of CD4 T cell help because none of these qualities were affected when memory CD8 T cells were transferred and maintained in either MHC class II- or CD4-deficient recipients.

Early and sustained innate immune response defines pathology and death in nonhuman primates infected by highly pathogenic influenza virus.

The mechanisms responsible for the virulence of the highly pathogenic avian influenza (HPAI) and of the 1918 pandemic influenza virus in humans remain poorly understood. To identify crucial components of the early host response during these infections by using both conventional and functional genomics tools, we studied 34 cynomolgus macaques (Macaca fascicularis) to compare a 2004 human H5N1 Vietnam isolate with 2 reassortant viruses possessing the 1918 hemagglutinin (HA) and neuraminidase (NA) surface proteins, known conveyors of virulence. One of the reassortants also contained the 1918 nonstructural (NS1) protein, an inhibitor of the host interferon response. Among these viruses, HPAI H5N1 was the most virulent. Within 24 h, the H5N1 virus produced severe bronchiolar and alveolar lesions. Notably, the H5N1 virus targeted type II pneumocytes throughout the 7-day infection, and induced the most dramatic and sustained expression of type I interferons and inflammatory and innate immune genes, as measured by genomic and protein assays. The H5N1 infection also resulted in prolonged margination of circulating T lymphocytes and notable apoptosis of activated dendritic cells in the lungs and draining lymph nodes early during infection. While both 1918 reassortant viruses also were highly pathogenic, the H5N1 virus was exceptional for the extent of tissue damage, cytokinemia, and interference with immune regulatory mechanisms, which may help explain the extreme virulence of HPAI viruses in humans.