PD-1 directed immunotherapy alters Tfh and humoral immune responses to seasonal influenza vaccine.

Anti-programmed death-1 (anti-PD-1) immunotherapy reinvigorates CD8 T cell responses in patients with cancer but PD-1 is also expressed by other immune cells, including follicular helper CD4 T cells (Tfh) which are involved in germinal centre responses. Little is known, however, about the effects of anti-PD-1 immunotherapy on noncancer immune responses in humans. To investigate this question, we examined the impact of anti-PD-1 immunotherapy on the Tfh-B cell axis responding to unrelated viral antigens. Following influenza vaccination, a subset of adults receiving anti-PD-1 had more robust circulating Tfh responses than adults not receiving immunotherapy. PD-1 pathway blockade resulted in transcriptional signatures of increased cellular proliferation in circulating Tfh and responding B cells compared with controls. These latter observations suggest an underlying change in the Tfh-B cell and germinal centre axis in a subset of immunotherapy patients. Together, these results demonstrate dynamic effects of anti-PD-1 therapy on influenza vaccine responses and highlight analytical vaccination as an approach that may reveal underlying immune predisposition to adverse events.

The Effect of CpG Sequences on Capsid-Specific CD8+ T Cell Responses to AAV Vector Gene Transfer.

Transfer of genes by adeno-associated virus (AAV) vectors is benefiting patients with particular genetic defects. Challenges remain by rejection of AAV-transduced cells, which may be caused by CD8+ T lymphocytes directed to AAV capsid antigens. Reducing the number of CpG motifs from the genome of AAV vectors reduces expansion of naive T cells directed against an epitope within the capsid. In contrast, AAV capsid-specific memory CD8+ T cells respond more vigorously to AAV vectors lacking CpG motifs than to those with CpG motifs presumably reflecting dampening of T cell expansion by cytokines from the innate immune system. Depending on the purification method, AAV vector preparations can contain substantial amounts of empty AAV particles that failed to package the genome. Others have used empty particles as decoys to AAV-neutralizing antibodies. We tested if empty AAV vectors given alone or mixed with genome-containing AAV vectors induce proliferation of naive or memory CD8+ T cells directed to an antigen within an AAV capsid. Naive CD8+ T cells failed to respond to empty AAV vectors, which in contrast induced expansion of AAV-specific memory CD8+ T cells.

Retraction Note: Engineered Polyallylamine Nanoparticles for Efficient In Vitro Transfection.

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1007/s11095-020-02971-0.

Safety and immunogenicity of a potential checkpoint blockade vaccine for canine melanoma.

Human immunotherapy with checkpoint blockades has achieved significant breakthroughs in recent years. In this study, a checkpoint blockade vaccine for canine melanoma was tested for safety and immunogenicity. Five healthy adult dogs received a mixture of three replication-defective chimpanzee-derived adenoviral vectors, one expressing mouse fibroblast-associated protein (mFAP) and the others expressing canine melanoma-associated antigens Trp-1 or Trp-2 fused into Herpes Simplex-1 glycoprotein D, a checkpoint inhibitor of herpes virus entry mediator (HVEM) pathways. The vaccine mixture was shown to be well tolerated and increased frequencies of canineTrp-1-specific activated CD8+ and CD4+ T cells secreting interferon-(IFN)-γ, tumor necrosis factor (TNF)-α, or interleukin (IL)-2 alone or in combinations in four and five out of five dogs, respectively. To avoid excessive bleeds, responses to cTrp-2 were not analyzed. All dogs responded with increased frequencies of mFAP-specific activated CD8+ and CD4+ T cells. The results of this safety/immunogenicity trial invite further testing of this checkpoint blockade vaccine combination in dogs with melanoma.

Circulating CXCR5+PD-1+ response predicts influenza vaccine antibody responses in young adults but not elderly adults.

Although influenza vaccination is recommended for all adults annually, the incidence of vaccine failure, defined as weak or absent increase in neutralizing Ab titers, is increased in the elderly compared with young adults. The T follicular helper cell (Tfh) subset of CD4 T cells provides B cell help in germinal centers and is necessary for class-switched Ab responses. Previous studies suggested a role for circulating Tfh cells (cTfh) following influenza vaccination in adults, but cTfh have not been studied in elderly adults in whom weak vaccine responses are often observed. In this study, we studied cTfh expressing CXCR5 and programmed death-1 (PD-1). cTfh from elderly adults were present at reduced frequency, had decreased in vitro B cell help ability, and had greater expression of ICOS compared with young adults. At 7 d after inactivated influenza vaccination, cTfh correlated with influenza vaccine-specific IgM and IgG responses in young adults but not in elderly adults. In sum, we have identified aging-related changes in cTfh that correlated with reduced influenza vaccine responses. Future rational vaccine design efforts should incorporate Tfh measurement as an immune correlate of protection, particularly in the setting of aging.

Vaccine-induced ICOS+CD38+ circulating Tfh are sensitive biosensors of age-related changes in inflammatory pathways.

Humoral immune responses are dysregulated with aging, but the cellular and molecular pathways involved remain incompletely understood. In particular, little is known about the effects of aging on T follicular helper (Tfh) CD4 cells, the key cells that provide help to B cells for effective humoral immunity. We performed transcriptional profiling and cellular analysis on circulating Tfh before and after influenza vaccination in young and elderly adults. First, whole-blood transcriptional profiling shows that ICOS+CD38+ cTfh following vaccination preferentially enriches in gene sets associated with youth versus aging compared to other circulating T cell types. Second, vaccine-induced ICOS+CD38+ cTfh from the elderly had increased the expression of genes associated with inflammation, including tumor necrosis factor-nuclear factor κB (TNF-NF-κB) pathway activation. Finally, vaccine-induced ICOS+CD38+ cTfh display strong enrichment for signatures of underlying age-associated biological changes. These data highlight the ability to use vaccine-induced cTfh as cellular "biosensors" of underlying inflammatory and/or overall immune health.

Comparative genomic study of spo0E family genes and elucidation of the role of Spo0E in Bacillus anthracis.

The propensity of bacterium to sporulate or retain the vegetative form depends on the amount of phosphorylated Spo0A (Spo0A(-P)), regulated by Spo0E multigene family of phosphatases (Spo0E, YisI and YnzD). Phylogenetic analysis revealed that Spo0E multigene family of phosphatases (SMFP) descends in two distinct clades of aerobic (Bacillus cluster) and anaerobic (Clostridia cluster) sporulating bacteria. High sequence conservation within species gives a notion that these members could have evolved through lineage and species-specific duplication event. Of the five genes in Bacillus cereus group, three are pathogen specific, and their synteny suggests that these paralogs could be involved in the regulation of amino acid metabolism and its transport. Overexpression of B. subtilis Spo0E, an ortholog of SMFP members in B. anthracis (BAS1251), resulted in sporulation deficient phenotype in B. anthracis. B. anthracis Spo0A(-P) binds to a consensus DNA sequence 5'-TGNCGAA-3' ('0A-like box') and loses its DNA binding ability following treatment with B. subtilis Spo0E. Thus, B. subtilis Spo0E acts on B. anthracis Spo0A(-P) and, therefore could complement the function of BAS1251. Further, since '0A-like box' are present in the promoter region of abrB gene, a known regulator of anthrax toxin gene expression, cross talk among SMFP members and Spo0A(-P)-AbrB could regulate the expression of anthrax toxin genes.

Age-related changes in B cell metabolism.

Antibody responses to vaccinations or infections decline upon aging. In this study we tested if metabolic changes in B cells may contribute to attenuation of responses to influenza vaccination in aged humans. Our data show that aging affects mitochondrial functions in B cells leading to increases in mitochondrial reactive oxygen species (MROS) and mitochondrial mass (MM) in some aged B cell subsets and decreases in expression levels of Sirtuin 1 (SIRT1), Forkhead box protein (FOX)O1 and carnitine palmitoyltransferase 1 (CPT-1). Seahorse analyses showed minor defects in glycolysis in the aged B cells after activation but a strong reduction in oxidative phosphorylation. The analyses of the transcriptome revealed further pronounced defects in one-carbon metabolism, a pathway that is essential for amino acid and nucleotide metabolism. Overall our data support the notion that the declining ability of aged B cells to increase their metabolism following activation contributes to the weakened antibody responses of the elderly.

Correlates of Protection Against SIVmac251 Infection in Rhesus Macaques Immunized With Chimpanzee-Derived Adenovirus Vectors.

We report on prime-boost vaccine regimens with two simian adenovirus (Ad) vectors (SAdV) or two human serotype Ad vectors (HAdV) expressing Gag and gp160 of simian immunodeficiency virus (SIV)mac239 tested in HAdV-seropositive rhesus macaques (RMs) repeatedly challenged rectally with low doses of SIVmac251. Both vaccine regimens reduced set point and peak viral loads (PVL) and accelerated viral clearance. In SAdV-vaccinated controller genotype RMs resistance against infection correlated with levels of envelope (Env)-specific antibody (Ab) titers. In both vaccine groups CD8+T cells controlled viral loads (VL) upon infection. Circulating CD4+ and CD8+ T cells showed significant changes in their transcriptome over time following vaccination, which differed between the vaccine groups. T cells from SIV-resistant RMs had unique transcriptional profiles indicating that both follicular T helper (TFH) cell responses and highly activated CD8+ T cells may play a role in protection.

The effect of timing of influenza vaccination and sample collection on antibody titers and responses in the aged.

Antibody responses, B cell subset distribution in blood and the blood transcriptome were analyzed in younger and aged human subjects before and after vaccination with the inactivated influenza vaccine. In the aged, but not the younger, individuals we saw a clear difference in antibody titers including those at baseline depending on the time of vaccination and sample collection. Differences in baseline titers in aged individuals treated in the morning or afternoon in turn affected responsiveness to the vaccine. In both younger and aged individuals, the time of sample collection also affected relative numbers of some of the B cell subsets in blood. A global gene expression analysis with whole blood samples from the aged showed small but statistically significant differences depending on the time of sample collection. Our data do not indicate that timing of vaccination affects immune responsiveness of the aged, but rather shows that in clinical influenza vaccine trials timing of collection of samples can have a major and potentially misleading influence on study outcome. In future vaccine trials, timing of vaccination and sample collection should be recorded carefully to allow for its use as a study covariant.

A Partial E3 Deletion in Replication-Defective Adenoviral Vectors Allows for Stable Expression of Potentially Toxic Transgene Products.

Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable glycoprotein 140 (gp140)- and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors.

A shortened interval between vaccinations with the trivalent inactivated influenza vaccine increases responsiveness in the aged.

We tested antibody responses to the trivalent inactivated influenza vaccine (TIV) in 34 aged individuals (>65 yrs) during the 2012/13 vaccination seasons. Nearly all had been vaccinated the previous year although the time interval between the two vaccine doses differed. One subgroup was re-vaccinated in 2012/13 within 6-9 months of their 2011/12 vaccination, the other received the two doses of vaccine in the typical ~12 month interval. Unexpectedly the sub-cohort with early revaccination exhibited significantly increased response rates and antibody titers to TIV compared to their normally re-vaccinated aged counter parts. Microarray analyses of gene expression in whole blood RNA taken at the day of the 2012/13 re-vaccination revealed statistically significant differences in expression of 754 genes between the individuals with early re-vaccination compared to subjects vaccinated in a normal 12 month interval. These observations suggest that TIV has long-lasting effects on the immune system affecting B cell responses as well as the transcriptome of peripheral blood mononuclear cells and this residual effect may augment vaccination response in patients where the effect of the previous vaccination has not yet diminished.

BTLA expression declines on B cells of the aged and is associated with low responsiveness to the trivalent influenza vaccine.

Virus-neutralizing antibody and B cell responses to influenza A viruses were measured in 35 aged and 28 middle-aged individuals following vaccination with the 2012 and 2013 trivalent inactivated influenza vaccines. Antibody responses to the vaccine strains were lower in the aged. An analysis of B cell subsets by flow cytometry with stains for immunoregulators showed that B cells of multiple subsets from the aged as compared to younger human subjects showed differences in the expression of the co-inhibitor B and T lymphocyte attenuator (BTLA). Expression of BTLA inversely correlated with age and appears to be linked to shifting the nature of the response from IgM to IgG. High BTLA expression on mature B cells was linked to higher IgG responses to the H1N1 virus. Finally, high BTLA expression on isotype switched memory B cells was linked to better preservation of virus neutralizing antibody titers and improved recall responses to vaccination given the following year.

Conformational fluctuations in anthrax protective antigen: a possible role of calcium in the folding pathway of the protein.

Protective antigen (PA) is the central receptor binding component of anthrax toxin, which translocates catalytic components of the toxin into the cytosol of mammalian cells. Ever since the crystal structure of PA was solved, there have been speculations regarding the possible role of calcium ions present in domain I of the protein. We have carried out a systematic study to elucidate the effect of calcium removal on the structural stability of PA using various optical spectroscopic techniques, limited proteolysis and mutational analysis. Urea denaturation studies clearly suggest that the unfolding pathway of the protein follows a non-two state transition with apo-PA being an intermediate species, whereas the folding pathway shows that calcium ions may be critical for the initial protein assembly.

Construction and characterization of E1- and E3-deleted adenovirus vectors expressing two antigens from two separate expression cassettes.

Here we describe a series of replication-defective adenovirus vectors designed to express transgene products from two expression cassettes placed into the deleted E1 and E3 domains. Vectors that contained an E1 cassette with a cytomegalovirus promoter in the forward orientation and an E3 cassette with the chicken ß-actin promoter in the reverse orientation grew to acceptable yields and expressed both transgenes. Additionally, they elicited immune responses to both transgene products. Levels of expression and the vectors' immunogenicity were influenced by the presence of regulatory elements shared between the two expression cassettes. Specifically, vectors that carried the same intron and enhancer in both expression cassettes could be rescued and expanded, but they were poorly immunogenic. Deletion of the enhancer or both the enhancer and the intron from the E3 cassette increased T- and B-cell responses to both transgene products.

B cell responses to the 2011/12-influenza vaccine in the aged.

Antibody and B cell responses to influenza A viruses were measured over a period of 2 months in 30 aged and 15 middle-aged individuals following vaccination with the 2011/12 trivalent inactivated influenza vaccine by micro-neutralization assays, ELISAs, ELISpot assays and cell surface staining with lineage-defining antibodies followed by multicolor flow cytometry. Both cohorts developed comparable antibody responses to the H3N2 virus of the vaccine while responses to the H1N1 virus were compromised in the aged. ELISpot assays of peripheral blood mononuclear cells (PBMCs) gave comparable results for the two cohorts. Analysis by flow cytometry upon staining of CD19+IgD-CD20- PBMCs with antibodies to CD27 and CD38 showed markedly reduced increases of such cells following vaccination in the aged. Additional analysis of cells from a subset of 10 younger and 10 aged individuals indicated that in the aged a portion of IgG producing cells lose expression of CD27 and reduce expression of CD38.

Vaccine-induced boosting of influenza virus-specific CD4 T cells in younger and aged humans.

Current yearly influenza virus vaccines induce strain-specific neutralizing antibody (NAb) responses providing protective immunity to closely matched viruses. However, these vaccines are often poorly effective in high-risk groups such as the elderly and challenges exist in predicting yearly or emerging pandemic influenza virus strains to include in the vaccines. Thus, there has been considerable emphasis on understanding broadly protective immunological mechanisms for influenza virus. Recent studies have implicated memory CD4 T cells in heterotypic immunity in animal models and in human challenge studies. Here we examined how influenza virus vaccination boosted CD4 T cell responses in younger versus aged humans. Our results demonstrate that while the magnitude of the vaccine-induced CD4 T cell response and number of subjects responding on day 7 did not differ between younger and aged subjects, fewer aged subjects had peak responses on day 14. While CD4 T cell responses were inefficiently boosted against NA, both HA and especially nucleocaspid protein- and matrix-(NP+M) specific responses were robustly boosted. Pre-existing CD4 T cell responses were associated with more robust responses to influenza virus NP+M, but not H1 or H3. Finally pre-existing strain-specific NAb decreased the boosting of CD4 T cell responses. Thus, accumulation of pre-existing influenza virus-specific immunity in the form of NAb and cross-reactive T cells to conserved virus proteins (e.g. NP and M) over a lifetime of exposure to infection and vaccination may influence vaccine-induced CD4 T cell responses in the aged.

Nanoparticles of cationic chimeric peptide and sodium polyacrylate exhibit striking antinociception activity at lower dose.

The current study investigates the performance of polyelectrolyte complexes based nanoparticles in improving the antinociceptive activity of cationic chimeric peptide-YFa at lower dose. Size, Zeta potential and morphology of the nanoparticles were determined. Size of the nanoparticles decreases and zeta potential increases with concomitant increase in charge ratio (Z(+/-)). The nanoparticles at Z(+/-)12 are spherical with 70+/-7 nm diameter in AFM and displayed positive surface charge and similar sizes (83+/-8 nm) by Zetasizer. The nanoparticles of Z(+/-) 12 are used in this study. Cytotoxicity by MTT assay on three different mammalian cell lines (liver, neuronal and kidney) revealed lower toxicity of nanoparticles. Hematological parameters were also not affected by nanoparticles compared to normal counts of water treated control group. Nanoparticles containing 10 mg/kg YFa produced increased antinociception, approximately 36%, in tail-flick latency test in mice, whereas the neat peptide at the same concentration did not show any antinociception activity. This enhancement in activity is attributed to the nanoparticle associated protection of peptide from proteolytic degradation. In vitro peptide release study in plasma also supported the antinociception profile of nanoparticles. Thus, our results suggest of a potential nanoparticle delivery system for cationic peptide drug candidates for improving their stability and bioavailability.

Properties of Bacillus anthracis spores prepared under various environmental conditions.

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45 degrees C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45 degrees C by wet heat, 2 M HCl, 2 M NaOH and 2 M H(2)O(2) was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45 degrees C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45 degrees C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45 degrees C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.

A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles.

Delivery of DNA and siRNA into mammalian cells is a powerful technique in treating various diseases caused by single gene defects. Herein, we report a highly efficient delivery system using 1,4-butanediol diglycidyl ether (bisepoxide) crosslinked polyethylenimine (PEI) nanoparticles (PN). The nanoparticle/DNA complexes (nanoplexes) exibited approximately 2.5- to 5.0-fold gene transfer efficacy and decreased cytotoxicity in cultured cell lines, compared to the native PEI (25 kDa) (gold standard) and commercially available transfection agents such as Lipofectamine 2000 and Fugene. The bisepoxide crosslinking results in change in amine ratio in PEI; however, it retains the net charge on PN unaltered. A series of nanoparticles obtained by varying the degree of crosslinking was found to be in the size range of 69-77 nm and the zeta potential varying from +35 to 40 mV. The proposed system was also found to deliver siRNA efficiently into HEK cells, resulting in approximately 70% suppression of the targetted gene (GFP).