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.

Immune response to influenza vaccination in the elderly is altered by chronic medication use.

BACKGROUND: The elderly patient population is the most susceptible to influenza virus infection and its associated complications. Polypharmacy is common in the aged, who often have multiple co-morbidities. Previous studies have demonstrated that commonly used prescription drugs can have extensive impact on immune defenses and responses to vaccination. In this study, we examined how the dynamics of immune responses to the two influenza A virus strains of the trivalent inactivated influenza vaccine (TIV) can be affected by patient's history of using the prescription drugs Metformin, NSAIDs or Statins. RESULTS: We provide evidence for differential antibody (Ab) production, B-cell phenotypic changes, alteration in immune cell proportions and transcriptome-wide perturbation in individuals with a history of long-term medication use, compared with non-users. We noted a diminished response to TIV in the elderly on Metformin, whereas those on NSAIDs or Statins had higher baseline responses but reduced relative increases in virus-neutralizing Abs (VNAs) or Abs detected by an enzyme-linked immunosorbent assay (ELISA) following vaccination. CONCLUSION: Collectively, our findings suggest novel pathways that might underlie how long-term medication use impacts immune response to influenza vaccination in the elderly. They provide a strong rationale for targeting the medication-immunity interaction in the aged population to improve vaccination responses.

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.

Immunological and virological analyses of rhesus macaques immunized with chimpanzee adenoviruses expressing the simian immunodeficiency virus Gag/Tat fusion protein and challenged intrarectally with repeated low doses of SIVmac.

Human adenovirus (AdHu)-based candidate AIDS vaccine can provide protection from simian immunodeficiency virus (SIV) transmission and disease progression. However, their potential use may be limited by widespread preexisting immunity to the vector. In contrast, preexisting immunity to chimpanzee adenoviruses (AdC) is relatively rare. In this study, we utilized two regimens of prime-boost immunizations with AdC serotype SAd-V23 (also called AdC6) and SAd-V24 (also called AdC7) expressing SIV Gag/Tat to test their immunogenicity and ability to protect rhesus macaques (RMs) from a repeated low-dose SIVmac239 challenge. Both AdC6 followed by AdC7 (AdC6/7) and AdC7 followed by AdC6 (AdC7/6) induced robust SIV Gag/Tat-specific T cell responses as measured by tetramer staining and functional assays. However, no significant protection from SIV transmission was observed in either AdC7/6- or AdC7/6-vaccinated RMs. Interestingly, in the RMs showing breakthrough infections, AdC7/6-SIV immunization was associated with a transient but significant (P = 0.035 at day 90 and P = 0.033 at day 120 postinfection) reduction in the setpoint viral load compared to unvaccinated controls. None of the measured immunological markers (i.e., number or functionality of SIV-specific CD8(+) and CD4(+) T cell responses and level of activated and/or CCR5(+) CD4(+) target cells) at the time of challenge correlated with protection from SIV transmission in the AdC-SIV-vaccinated RMs. The robust immunogenicity observed in all AdC-immunized RMs and the transient signal of protection from SIV replication exhibited by AdC7/6-vaccinated RMs even in the absence of any envelope immunogen suggest that AdC-based vectors may represent a promising platform for candidate AIDS vaccines.

Hexon-modified recombinant E1-deleted adenovirus vectors as dual specificity vaccine carriers for influenza virus.

To determine if an ordered and repetitive display of an epitope promoted induction of superior antibody responses, we compared B-cell responses to an influenza A virus epitope that was either encoded as a transgene by an adenovirus (Ad) vector or expressed on the vector's surface. To this end, we constructed a panel of influenza A virus vaccines based on chimpanzee-derived replication-defective adenovirus (AdC) vectors of serotype SAd-V25 also called AdC68. AdC68 vectors were modified to express a linear B-cell epitope of the ectodomain of matrix 2 (M2e) within variable regions 1 (VR1) or 4 (VR4) of the adenovirus hexon. Additional vectors with wild-type or M2e-modified hexon encoded M2e fused to the influenza A virus nucleoprotein (NP) as a transgene product. Hexon-modified vectors were tested for immunogenicity and efficacy in mice in comparison to vectors with native hexon expressing the M2e-NP fusion protein. Upon priming, vectors expressing M2e within VR1 of hexon induced M2e-specific antibody responses of higher magnitude and avidity than those carrying M2e within VR4 or vectors expressing the M2e as part of a transgene product. CD8(+) T-cell responses to the transgenic NP were comparable between vectors. M2e-specific antibody responses could be boosted by a second dose of the VR1 hexon-modified vector but not by repeated immunization with the VR4 hexon-modified vector.

Treatment with the PPARα agonist fenofibrate improves the efficacy of CD8+ T cell therapy for melanoma.

Adoptive transfer of tumor antigen-specific CD8+ T cells can limit tumor progression but is hampered by the T cells' rapid functional impairment within the tumor microenvironment (TME). This is in part caused by metabolic stress due to lack of oxygen and glucose. Here, we report that fenofibrate treatment of human ex vivo expanded tumor-infiltrating lymphocytes (TILs) improves their ability to limit melanoma progression in a patient-derived xenograft (PDX) mouse model. TILs treated with fenofibrate, a peroxisome proliferator receptor alpha (PPARα) agonist, switch from glycolysis to fatty acid oxidation (FAO) and increase the ability to slow the progression of autologous melanomas in mice with freshly transplanted human tumor fragments or injected with tumor cell lines established from the patients' melanomas and ex vivo expanded TILs.

Vaccine-induced T cells provide partial protection against high-dose rectal SIVmac239 challenge of rhesus macaques.

Despite enormous efforts by the scientific community, an effective HIV vaccine remains elusive. To further address to what degree T cells in absence of antibodies may protect against simian immunodeficiency virus (SIV) disease progression, rhesus macaques were vaccinated intramuscularly with a chimpanzee-derived Ad vector (AdC) serotype 6 and then boosted intramuscularly with a serologically distinct AdC vector of serotype 7 both expressing Gag of SIVmac239. Animals were subsequently boosted intramuscularly with a modified vaccinia Ankara (MVA) virus expressing Gag and Tat of the homologous SIV before mucosal challenge with a high dose of SIVmac239 given rectally. Whereas vaccinated animals showed only a modest reduction of viral loads, their overall survival was improved, in association with a substantial protection from the loss of CD4(+) T cells. In addition, the two vaccinated Mamu-A*01(+) macaques controlled viral loads to levels below detection within weeks after challenge. These data strongly suggest that T cells, while unable to affect SIV acquisition upon high-dose rectal infection, can reduce disease progression. Induction of potent T-cell responses should thus remain a component of our efforts to develop an efficacious vaccine to HIV-1.

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.

Cross-linked polyethylenimine-hexametaphosphate nanoparticles to deliver nucleic acids therapeutics.

Branched polyethylenimine (PEI; 25 kDa) as a nonviral vector exhibits high transfection efficiency and is a potential candidate for efficient gene delivery. However, the cytotoxicity of PEI limits its application in vivo. PEI was ionically interacted with hexametaphosphate, a compact molecule with high anionic charge density, to obtain nanoparticles (PEI-HMP). Nanoparticles were assessed for their efficacy in protecting complexed DNA against nucleases. The intracellular trafficking of nanoparticles was monitored by confocal microscopy. The cytotoxicity and transfection efficiency of PEI-HMP nanoparticles were evaluated in vitro. In vitro transfection efficiency of PEI-HMP (7.7%) was approximately 1.3- to 6.4-folds higher than that of the commercial reagents GenePORTER 2, Fugene, and Superfect. Also, PEI-HMP (7.7%) delivered green fluorescent protein (GFP)-specific small interfering ribonucleic acid (siRNA) in culture cells leading to >80% suppression in GFP gene expression. PEI-HMP nanoparticles protected complexed DNA against DNase for at least 2 hours. A time-course uptake of PEI-HMP (7.7%) nanoparticles showed the internalization of nanoparticles inside the cell nucleus in 2 hours. Thus, PEI-HMP nanoparticles efficiently transfect cells with negligible cytotoxicity and show great promise as nonviral vectors for gene delivery. FROM THE CLINICAL EDITOR: Branched polyethylenimine (PEI) as a non-viral vector exhibits high transfection efficiency for gene delivery, but its cytotoxicity limits its applications. PEI hexametaphosphate nanoparticles (PEI-HMP) demonstrated a 1.3-6.4 folds higher transfection rate compared to commercial reagents. Overall, PEI-HMP nanoparticles efficiently transfect cells with negligible cytotoxicity and show great promise as non-viral vectors for gene delivery.

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.

Enhancing CD8+ T Cell Fatty Acid Catabolism within a Metabolically Challenging Tumor Microenvironment Increases the Efficacy of Melanoma Immunotherapy.

How tumor-infiltrating T lymphocytes (TILs) adapt to the metabolic constrains within the tumor microenvironment (TME) and to what degree this affects their ability to combat tumor progression remain poorly understood. Using mouse melanoma models, we report that CD8+ TILs enhance peroxisome proliferator-activated receptor (PPAR)-α signaling and catabolism of fatty acids (FAs) when simultaneously subjected to hypoglycemia and hypoxia. This metabolic switch partially preserves CD8+ TILs' effector functions, although co-inhibitor expression increases during tumor progression regardless of CD8+ TILs' antigen specificity. Further promoting FA catabolism improves the CD8+ TILs' ability to slow tumor progression. PD-1 blockade delays tumor growth without changing TIL metabolism or functions. It synergizes with metabolic reprogramming of T cells to achieve superior antitumor efficacy and even complete cures.

Age-related changes in the transcriptome of antibody-secreting cells.

We analyzed age-related defects in B cell populations from young and aged mice. Microarray analysis of bone marrow resident antibody secreting cells (ASCs) showed significant changes upon aging, affecting multiple genes, pathways and functions including those that play a role in immune regulation, humoral immune responses, chromatin structure and assembly, cell metabolism and the endoplasmic reticulum (ER) stress response. Further analysis showed upon aging defects in energy production through glucose catabolism with reduced oxidative phosphorylation. In addition aged B cells had increased levels of reactive oxygen-species (ROS), which was linked to enhanced expression of the co-inhibitor programmed cell death (PD)-1.

Race-related differences in antibody responses to the inactivated influenza vaccine are linked to distinct pre-vaccination gene expression profiles in blood.

We conducted a 5-year study analyzing antibody and B cell responses to the influenza A virus components of the inactivated influenza vaccine, trivalent (IIV3) or quadrivalent (IIV4) in younger (aged 35-45) and aged (≥65 years of age) Caucasian and African American individuals. Antibody titers to the two influenza A virus strains, distribution of circulating B cell subsets and the blood transcriptome were tested at baseline and after vaccination while expression of immunoregulatory markers on B cells were analyzed at baseline. African Americans mounted higher virus neutralizing and IgG antibody responses to the H1N1 component of IIV3 or 4 compared to Caucasians. African Americans had higher levels of circulating B cell subsets compared to Caucasians. Expression of two co-regulators, i.e., programmed death (PD)-1 and the B and T cell attenuator (BTLA) were differentially expressed in the two cohorts. Race-related differences were caused by samples from younger African Americans, while results obtained with samples of aged African Americans were similar to those of aged Caucasians. Gene expression profiling by Illumina arrays revealed highly significant differences in 1368 probes at baseline between Caucasians and African Americans although samples from both cohorts showed comparable changes in transcriptome following vaccination. Genes differently expressed between samples from African Americans and Caucasians regardless of age were enriched for myeloid genes, while the transcripts that differed in expression between younger African Americans and younger Caucasians were enriched for those specific for B-cells.

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.