Analysis Of Cellular Immune Responses Research Articles

The Pathogenesis of Mesh-induced Inflammatory Response and Pain: Rationale for Development of New Mesh

Chronic postoperative pain (CPP) in mesh hernia repair (MHR) may complicate the postoperative course. The cause of CPP may be multifactorial - surgical technique, patient-intrinsic factors, and mesh. Polypropylene (PP) mesh is the most widely used material for MHR. Despite its advantages, it has been associated with severe complications in urogynecology leading to a partial mesh ban. PP is not inert and causes foreign body reactions (FBR), corrosion, and loss in biocompatibility. Pain is a hallmark of mesh-induced complications. The pathogenesis of pain is related to an immune response with neutrophils, T cells, and macrophages, major players in mesh-associated fibrosis and pain. Pain may be caused by mesh implantation-induced nerve entrapment, compression, and severe inflammation, relevant for both nociceptive and neuropathic pain. Compression neuropathy has been associated with preoperative pain and chronic postoperative pain in mesh and non-mesh repairs. The mesh may induce FBR changes causing clinical complications and pain. Increased mesh vicinity innervation induced by fibrosis may be responsible for chronic postoperative pain. An aggressive immune response in pelvic floor reconstructive surgery degrades PP. T cells and macrophages may protect against or induce degradation and pain. The main point to eliminate pain is to develop a mesh, that provides long-term corrosion resistance, and biocompatibility. This may be achieved by coating PP mesh with a thin layer of Titanium oxide or meshes of pure titanium. Titanium is considered to be bio-inert providing corrosion resistance and biocompatibility. However, depending on the location and surface of the mesh (roughness, hydrophilicity), there may be a macrophage-neutrophil-induced inflammatory response causing fibrosis and cicatrization. Based on the structure, location, and production Titanium may demonstrate beneficial effects concerning corrosion, oxidation, FBR, and biocompatibility. To improve outcomes in MHR the analysis of cellular immune response concerning mesh properties, composite endpoints, pain, and physical function may be necessary.

The BD Rhapsody&[trade] HT Xpress System supports dynamic and high-throughput single-cell capture enabling a seamless workflow for analysis of cellular immune responses

Abstract The BD Rhapsody™ Single-Cell Analysis System is an innovative platform that aids high-throughput sequencing of mRNA and proteins in thousands of single cells. Here we present a new eight-lane cartridge design concept, the BD Rhapsody™ HT Xpress System. This novel device architecture can reduce both hands-on and assay time by enabling the users to simultaneously load multiple cell samples while preserving the flexibility to use one lane at a time. To demonstrate the increased throughput capabilities and workflow flexibility of the system, we measured in vitro T cell activation over time. To do this, peripheral blood mononuclear cells from four different subjects were prepared for cell culture in IL-2 or IL-2/anti-CD3/anti-CD28. After one day in culture, cell samples from each culture condition were barcoded using the BD® Single-Cell Multiplexing Kit. Cells from the same subject were pooled and stained with the BD® AbSeq Immune Discovery Panel. Pooled cells from each subject were loaded into the cartridge (one subject per lane, four lanes total). Remaining cells were cultured for another day and similarly barcoded, stained and loaded into the other four lanes of the cartridge. Eight different next generation sequencing libraries were generated and analyzed with the BD Rhapsody™ Analysis Pipeline and SeqGeq™ Software. This strategy enabled us to assess dynamic changes in both T cell surface protein profiles and transcriptome during cell activation while using a streamlined workflow for concomitant analysis of multiple samples. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, BD Rhapsody and SeqGeq are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved.

Development of a Monoclonal Antibody to Pig CD69 Reveals Early Activation of T Cells in Pig after PRRSV and ASFV Infection.

The CD69 molecule, as an early activation marker of lymphocytes, is often used to assess the activation of cellular immunity. However, for pigs, an anti-pig CD69 antibody is not yet available for this purpose after infection or vaccination. In this study, a monoclonal antibody (mAb) against pig CD69 was produced by peptide immunization and hybridoma technique. One mAb (5F12) showed good reactivity with pig CD69 that was expressed in transfected-HEK-293T cells and on mitogen-activated porcine peripheral blood mononuclear cells (PBMCs) by indirect immunofluorescence assay and flow cytometry. This mAb did not cross-react with activated lymphocytes from mouse, bovine, and chicken. Epitope mapping showed that the epitope recognized by this mAb was located at amino acid residues 147–161 of pig CD69. By conjugating with fluorochrome, this mAb was used to detect the early activation of lymphocytes in PRRSV- and ASFV-infected pigs by flow cytometry. The results showed that PRRSV infection induced the dominant activation of CD4 T cells in mediastinal lymph nodes and CD8 T cells in the spleen at 14 days post-infection, in terms of CD69 expression. In an experiment on ASFV infection, we found that ASFV infection resulted in the early activation of NK cells, B cells, and distinct T cell subsets with variable magnitude in PBMCs, spleen, and submandibular lymph nodes. Our study revealed an early event of lymphocyte and T cell activation after PRRSV and ASFV infections and provides an important immunological tool for the in-depth analysis of cellular immune response in pigs after infection or vaccination.

Transcriptome-wide analysis of cellular immune response stimulated by nuclear input of different down syndrome cell adhesion molecule intracellular domains

In arthropods, Dscam (Down syndrome cell adhesion molecule) produces multiple pathogen specific receptors via immune responsive alternative splicing, generating molecular complexity analogous to vertebrate antibodies. Fewer isoforms are produced by the exons encoding Dscam's intracellular domain (ICD); therefore, the present study aimed to determine the transcriptional response of Eriocheir sinensis to Dscam ICDs. In the group overexpressing all cytoplasmic tail exons (ICD-FL), 1401 differentially expressed genes (DEGs) were identified; overexpressed of ICD constructs lacking exon-35 (ICD-△35) identified 413 DEGs; and overexpression of ICD constructs lacking exon-35 and exon-36 (ICD-△35 + 36) identified 22 DEGs. The DEGs were enriched in immunity and metabolism-related pathways. The expression of selected genes was confirmed using quantitative real-time reverse transcription PCR. The transcriptomes of Drosophila S2 cells overexpressing different ICDs were then determined. We identified key immune, metabolic, and cell proliferation-regulated genes and gene networks, providing insights into the membrane-to-nuclear signaling pathway of Dscam.

Interim Analysis of Immunogenicity to the Vector Capsid and Transgene-Expressed Human FVIII in a Phase-1/2 Clinical Study of Bmn 270, an AAV5-Mediated Gene Therapy for Hemophilia a

▪When assessing the immunogenicity of gene therapy drug products, two components need to be considered: immunogenicity toward the vector delivery system and immunogenicity specific for the transgene product. BMN 270 is an AAV5-mediated gene therapy indicated for the treatment of Hemophilia A, and encodes for a codon-optimized B-domain deleted human FVIII protein (hFVIII-SQ) under control of a liver specific promoter. In study BMN 270-201, the first-in-human clinical trial, prospective patients with severe Hemophilia A were screened for both pre-existing antibodies directed against the AAV5 vector and inhibitors of transduction using a cell-based in vitro transduction inhibition (TI) assay. Patients testing positive in either assay were excluded from the trial, as were patients who had a history of FVIII inhibitors. Following infusion of BMN 270, patient plasma was analyzed for total antibody (TAb) responses specific for the AAV5 capsid and TAb responses directed against FVIII, using bridging ECLA immunoassays. Development of FVIII inhibitors was monitored using the Nijmegen-modified Bethesda assay. Additionally, peripheral blood mononuclear cells were collected for analysis by IFN-γ and TNF-α ELISpot assays for detection of capsid-specific and hFVIII-SQ specific cellular immune responses. The available data indicate that, as expected, all patients develop anti-AAV5 TAb by Week 8 post BMN 270 infusion, the first immunogenicity time-point assessed. One patient screened and confirmed positive at a single time point in the anti-FVIII TAb assay. This response was below the minimum required dilution to determine a titer, and was negative at all subsequent time points. No patients have tested positive in the Bethesda assay for FVIII inhibitors.We investigated the possibility that cell mediated immunity may explain the observed elevation in hepatic enzymes. To date, several patients dosed with BMN 270 experienced asymptomatic elevation of alanine aminotransferase (ALT) laboratory values ranging from 44 to 141 U/L (normal, 6-43 U/L) that resolved without sequelae. Analysis of cellular immune response in these patients by IFN-γ ELISpot assay against peptides spanning AAV5 capsid or the hFVIII-SQ protein was negative, as it was for all other subjects in the study across all time points tested to date. Unlike the IFN-γ assay, intermittent responses were detected across several patients in response to hFVIII-SQ peptide pools in the TNF-α ELISpot assay. In general, these responses were self-limiting and were not temporally associated with increases in ALT or a decline in FVIII activity measures, and did not correlate with results from the IFN-γ assay. Similar TNF-α responses to hFVIII-SQ peptides were also observed in a subset of healthy donors. Overall, immune responses to BMN 270 were characterized by the production of anti-AAV5 antibodies, which is an expected finding following viral vector administration. In conclusion, no consistent association could be made with increases in ALT and cellular immune responses. Our data to date indicate that although antibodies to the AAV5 capsid develop in all patients, concomitant cellular immunity is not readily detectable, and has not been associated with liver function abnormalities nor changes in transgene expression levels. DisclosuresLong:BioMarin: Employment. Kim:BioMarin: Employment. Wong:BioMarin: Employment. Yang:BioMarin: Employment. Vettermann:BioMarin: Employment. Pryer:BioMarin: Employment. Hardet:Genethon: Employment. Kuranda:Genethon: Employment. Mingozzi:Genethon: Employment. Pierce:Roche: Consultancy; BioMarin: Consultancy. Schweighardt:BioMarin: Employment.

Characterizing the cellular immune response to subretinal AAV gene therapy in the murine retina

Although adeno-associated viral (AAV) vector-mediated retinal gene therapies have demonstrated efficacy, the mechanisms underlying dose-dependent retinal inflammation remain poorly understood. Here, we present a quantitative analysis of cellular immune response to subretinal AAV gene therapy in mice using multicolor flow cytometry with a panel of key immune cell markers. A significant increase in CD45+ retinal leukocytes was detected from day 14 post-subretinal injection of an AAV8 vector (1 × 109 genome copies) encoding green fluorescent protein (GFP) driven by a ubiquitous promoter. These predominantly consisted of infiltrating peripheral leukocytes including macrophages, natural killer cells, CD4 and CD8 T cells, and natural killer T cells; no significant change in resident microglia population was detected. This cellular response was persistent at 28 days and suggestive of type 1 cell-mediated effector immunity. High levels (80%) of GFP fluorescence were found in the microglia, implicating their role in viral antigen presentation and peripheral leukocyte recruitment. When compared against AAV.GFP in paired eyes, an equivalent dose of an otherwise identical vector encoding the human therapeutic transgene Rab-escort protein 1 (REP1) elicited a significantly diminished cellular immune response (4.2-fold; p = 0.0221). However, the distribution of immune cell populations remained similar, indicating a common mechanism of AAV-induced immune activation.

Homologous prime-boost with Zika virus envelope protein and poly (I:C) induces robust specific humoral and cellular immune responses

The recent outbreaks of Zika virus (ZIKV) infection and the potential association with Guillain-Barré syndrome in adults and with congenital abnormalities have highlighted the urgency for an effective vaccine. The ZIKV Envelope glycoprotein (EZIKV) is the most abundant protein on the virus surface, and has been evaluated together with the pre-membrane protein (prM) of the viral coat as a vaccine candidate in clinical trials. In this study, we performed a head-to-head comparison of the immune response induced by different EZIKV-based vaccine candidates in mice. We compared different platforms (DNA, recombinant protein), adjuvants (poly (I:C), CpG ODN 1826) and immunization strategies (homologous, heterologous).The hierarchy of adjuvant potency showed that poly (I:C) was a superior adjuvant than CpG ODN. While poly (I:C) assisted immunization reached a plateau in antibody titers after two doses, the CpG ODN group required an extra immunization dose. Besides, the administration of poly (I:C) induced higher EZIKV-specific cellular immune responses than CpG ODN. We also show that immunization with homologous prime-boost EZIKV protein + poly (I:C) regimen induced a more robust humoral response than homologous DNA (pVAX-EZIKV) or heterologous regimens (DNA/protein or protein/DNA). A detailed analysis of cellular immune responses revealed that homologous (EZIKV + poly (I:C)) and heterologous (pVAX-EZIKV/EZIKV + poly (I:C)) prime-boost regimens induced the highest magnitude of IFN-γ secreting cells and cytokine-producing CD4+ T cells.Overall, our data demonstrate that homologous EZIKV + poly (I:C) prime-boost immunization is sufficient to induce more robust specific-EZIKV humoral and cellular immune responses than the other strategies that contemplate homologous DNA (pVAX-EZIKV) or heterologous (pVAX-EZIKV/EZIKV + poly (I:C), and vice-versa) immunizations.

Analyses of Cellular Immune Responses in Ferrets Following Influenza Virus Infection.

Ferrets are an ideal animal model in which to study the transmission of respiratory viruses as well as disease progression and vaccine efficacy because of their close anatomical and physiological resemblances to humans. However, a paucity of reagents and standardized procedures has hampered research progress, especially for studying cell-mediated immunity. The approaches described here-leukocyte isolation from whole blood and secondary lymphoid tissues-are generalizable, highly reproducible, and deliver single cell suspensions with excellent cell viability. Importantly, we have now developed assays to quantify key cellular components and antigen-specific T cell responses at the single cell level from multiple tissue compartments following influenza infection in ferrets. Collectively, these methods were instrumental in flow cytometry studies that revealed alterations in immune cell composition and distribution across lymphoid tissues following viral infection. Furthermore, sorting of T cell populations and peptide restimulation ex vivo in cytokine ELISpot assays has provided novel insight into the influenza-specific CD4 and CD8 T cell repertoire. The detailed procedures for these techniques are described in this chapter and can likely be adapted for the analyses of responses to many respiratory pathogens.

Retrospective Proteomic Analysis of Cellular Immune Responses and Protective Correlates of p24 Vaccination in an HIV Elite Controller Using Antibody Arrays.

Background: HIV p24 is an extracellular HIV antigen involved in viral replication. Falling p24 antibody responses are associated with clinical disease progression and their preservation with non-progressive disease. Stimulation of p24 antibody production by immunization to delay progression was the basis of discontinued p24 vaccine. We studied a therapy-naive HIV+ man from Sydney, Australia, infected in 1988. He received the HIV-p24-virus like particle (VLP) vaccine in 1993, and continues to show vigorous p24 antigen responses (>4% p24-specific CD4+ T cells), coupled with undetectable plasma viremia. We defined immune-protective correlates of p24 vaccination at the proteomic level through parallel retrospective analysis of cellular immune responses to p24 antigen in CD4+ and CD8+ T cells and CD14+ monocytes at viremic and aviremic phases using antibody-array. We found statistically significant coordinated up-regulation by all three cell-types with high fold-changes in fractalkine, ITAC, IGFBP-2, and MIP-1α in the aviremic phase. TECK and TRAIL-R4 were down-regulated in the viremic phase and up-regulated in the aviremic phase. The up-regulation of fractalkine in all three cell-types coincided with protective effect, whereas the dysfunction in anti-apoptotic chemokines with the loss of immune function. This study highlights the fact that induction of HIV-1-specific helper cells together with coordinated cellular immune response (p < 0.001) might be important in immunotherapeutic interventions and HIV vaccine development.

Comparative Analysis of Cellular Immune Responses in Treated Leishmania Patients and Hamsters against Recombinant Th1 Stimulatory Proteins of Leishmania donovani.

Our prior studies demonstrated that cellular response of T helper 1 (Th1) type was generated by a soluble antigenic fraction (ranging from 89.9 to 97.1 kDa) of Leishmania donovani promastigote, in treated Leishmania patients as well as hamsters and showed significant prophylactic potential against experimental visceral leishmaniasis (VL). Eighteen Th1 stimulatory proteins were identified through proteomic analysis of this subfraction, out of which 15 were developed as recombinant proteins. In the present work, we have evaluated these 15 recombinant proteins simultaneously for their comparative cellular responses in treated Leishmania patients and hamsters. Six proteins viz. elongation factor-2, enolase, aldolase, triose phosphate isomerase, protein disulfide isomerase, and p45 emerged as most immunogenic as they produced a significant lymphoproliferative response, nitric oxide generation and Th1 cytokine response in PBMCs and lymphocytes of treated Leishmania patients and hamsters respectively. The results suggested that these proteins may be exploited for developing a successful poly-protein and/or poly-epitope vaccine against VL.

High normal values of circulating immune cell subsets before surgery may be protective against development of postoperative acute kidney injury

Occlusion of renal artery during stenting procedure by endovascular aortic repair leads to ischemic-reperfusion injury (IRI). Additionally, significant amount of intravenous contrast agent is used during the procedure. Therefore acute kidney injury (AKI) is one of the most common postoperative complications. Current experimental data suggest an important role of systemic immune reaction in development of AKI after IRI and nephrotoxic agents. However, robust analysis of cellular immune response in clinical setting and its relation to AKI after IRI and iv contrast have not been previously reported.

Comparative analysis of cellular immune responses and cytokine levels in sheep experimentally infected with bluetongue virus serotype 1 and 8

Protective immunity in sheep with bluetongue virus (BTV) infection as well as the role of BTV-induced cytokines during immune response remains unclear. Understanding the basis immunological mechanisms in sheep experimentally infected with serotypes 1 and 8 (BTV-1 and -8) was the aim of this study. A time-course study was carried out in order to evaluate cell-mediated immune response and serum concentrations of cytokines (IL-1β, TNFα, IL-12, IFNγ, IL-4 and IL-10) with inflammatory and immunological functions. Depletion of T cell subsets (mainly CD4+, γδ and CD25+) together with the absence of cytokines (IFNγ and IL-12) involved in the regulation of cell-mediated antiviral immunity at the first stage of the disease suggested that both BTV-1 and BTV-8 might impair host's capability against primary infections which would favor viral replication and spreading. However, cellular immune response and cytokines elicited an immune response in sheep that efficiently reduced viremia in the final stage of the experiment. Recovery of T cell subsets (CD4+ and CD25+) together with a significant increase of CD8+ T lymphocytes in both infected groups were observed in parallel with the decrease of viremia. Additionally, the recovery of CD4+ T lymphocytes together with the significant increase of IL-4 serum levels at the final stage of the experiment might contribute to humoral immune response activation and neutralizing antibodies production against BTV previously described in the course of this experiment. These results suggested that both cellular and humoral immune response may contribute to protective immunity against BTV-1 and BTV-8 in sheep. The possible role played by IL-10 and CD25+ cells in controlling inflammatory and immune response in the final stage of the experiment has also been suggested.

Immunological basis of anti-Candida vaccines focused on synthetically prepared cell wall mannan-derived manno-oligomers.

The increasing incidence of diseases caused by Candida species and complications in individuals with impaired immunity require new strategies for candidiasis treatment and prevention. The available therapies are often of limited effectiveness in immunocompromised patients, resulting in treatment failures, chronic infections and high mortality rates. Research directed at identifying the composition of an effective vaccine is required. Mannan forms the outermost layer of the Candida cell wall and has an essential role in modulation of anti-Candida host immune responses. Therefore, Candida cell wall mannan and synthetically prepared manno-oligomer-based glycoconjugates are the foci of attention in vaccine candidate development. Almost all of the existing human vaccines mediate protection through neutralizing antibodies. Th1-based and/or Th17-based cellular immune responses, rather than antibody-mediated immunity, mediate protection against candidiasis. Findings of published studies indicate that analysis of cellular immune responses as well as antibody responses is necessary when assessing the immunomodulatory properties of manno-oligomer-based glycoconjugates that are potential anti-Candida vaccine candidates.

DNA vaccination by intradermal electroporation induces long-lasting immune responses in rhesus macaques.

BackgroundA desirable HIV vaccine should induce protective long-lasting humoral and cellular immune responses.MethodsMacaques were immunized by env DNA, selected from a panel of recently transmitted SIVmac251 Env using intradermal electroporation as vaccine delivery method and magnitude, breadth and longevity of humoral and cellular immune responses.ResultsThe macaques developed high, long-lasting humoral immune responses with neutralizing capacity against homologous and heterologous Env. The avidity of the antibody responses was also preserved over 1-year follow-up. Analysis of cellular immune responses demonstrated induction of Env-specific memory T cells harboring granzyme B, albeit their overall levels were low. Similar to the humoral responses, the cellular immunity was persistent over the ∼1-year follow-up.ConclusionThese data show that vaccination by this intradermal DNA delivery regimen is able to induce potent and durable immune responses in macaques.

The MultiTEP platform–based Alzheimer's disease epitope vaccine activates a broad repertoire of T helper cells in nonhuman primates

BackgroundAs a prelude to clinical trials we have characterized B- and T-cell immune responses in macaques to AD vaccine candidates: AV-1955 and its slightly modified version, AV-1959 (with 3 additional promiscuous Th epitopes). MethodsT- and B-cell epitope mapping was performed using the ELISPOT assay and competition ELISA, respectively. ResultsAV-1955 and AV-1959 did not stimulate potentially harmful autoreactive T cells, but instead activated a broad but individualized repertoire of Th cells specific to the MultiTEP platform in macaques. Although both vaccines induced robust anti-Aβ antibody responses without producing antibodies specific to Th epitopes of MultiTEP platforms, analyses of cellular immune responses in macaques demonstrated that the addition of Th epitopes in the case of AV-1959 created a more potent, superior vaccine. ConclusionAV-1959 is a promising vaccine candidate capable of producing therapeutically potent anti-amyloid antibody in a broader population of vaccinated subjects with high MHC class II gene polymorphisms.

Procedures for mucosal immunization and analyses of cellular immune response to candidate HIV vaccines in murine and nonhuman primate models.

Sampling the mucosal tissues and analyses of immune responses are integral to vaccine-development strategies against human immunodeficiency virus (HIV), which is transmitted predominantly across the oro-genital mucosa. While immune assay development and standardization attempts employ mouse models, immunogenicity and protective efficacy that can be extrapolated to humans are realized only from experiments in nonhuman primates. Here, we describe commonly used practices for immunizations in rhesus macaques (Macaca mulatta) along with procedures for obtaining important mucosal tissues samples from macaques and mice. We also describe detailed protocols for two important assays applicable in mouse as well as primate experiments for determining antigen-specific T cells responses induced after vaccination.

Retrospective proteomic analysis of cellular immune responses and protective correlates of p24 vaccination in an HIV elite controller

BackgroundHIV p24 is an extracellular HIV antigen that is involved in viral replication. Falling p24 antibody responses are associated with clinical disease progression, whereas their preservation with non-progression implies that stimulation of p24 antibody production by immunisation might delay progression. This was the basis of the discontinued p24 vaccine. We identified a therapy-naive man aged 61 years from Sydney, Australia, infected in 1988 through his partner. He received the HIV-p24-virus like particle (VLP) vaccine and a 3 week course of azidothymidin monotherapy in 1993, and continued to show vigorous p24 antigen responses with more than 4% p24-specific CD4+ T cells, coupled with undetectable plasma viraemia, until December, 2011. The patient has since remained therapy naive, with plasma viraemia below detectable levels. The patient is heterozygous for CCR5Δ32 and has an HLA type A1, 2; B8, 44; DR4, 15. In 2011, a transitory viral spike (163 HIV RNA copies per mL plasma) was recorded, which without antiretroviral drug treatment reverted to undetectable virus (<20 HIV copies per mL) in 3 weeks. We aimed to define the immune-protective correlates of p24 vaccination in this individual by retrospective analysis of cellular responses to p24 antigen in CD4+ and CD8+ T cells, and CD14+ monocytes, in terms of the concentrations of 270 cytokines and chemokines during viraemia and aviraemia. MethodsWe used whole peripheral blood mononuclear cells from the two phases, stimulated them with p24 antigen, and then separated them by magnetic beads as stimulated and unstimulated CD4+ and CD8+ T cells, and monocyte fractions. Cellular proteins were extracted, quantified, and analysed with a cytokine antibody array. Differentially expressed proteins were analysed with online bioinformatic tools, and statistics evaluated with the student t test and three k-way analyses. FindingsEncompassing all three cell types, we found statistically significant coordinated upregulation with high fold-changes of fractalkine, ITAC, IGFBP-2, cathepsin-S, and CCL14a in the aviraemic phase. TECK and TRAIL-R4 were downregulated in the viraemic phase and upregulated in the aviraemic phase. Fractalkine, which showed high fold-changes across cell types during the aviraemic phase and no expression during viraemia, plays a vital part in promotion of cell survival during homeostatic and inflammatory conditions. The upregulation of fractalkine in all three cell types (14-fold in CD4+ T cells, 12-fold in monocytes, and five-fold in CD8+ T cells) during aviraemia might be associated with a protective effect. Interestingly, downregulation of TRAIL-R3/R4 and TECK/CCL25 coincided with plasma viraemia and their upregulation with aviraemia, suggesting that dysfunction in antiapoptotic chemokines might be a mechanism contributing to loss of immune function during HIV infection, and that an effective T-cell-derived immune response can be mounted without T-cell exhaustion. Decrease in TECK expression is associated with increased apoptosis in lymphoid tissues in macaques infected with simian immunodeficiency virus. InterpretationBecause the p24 VLP vaccine failed to induce antibody titres, the possible beneficial effect of p24 vaccine in this individual could be attributed to broad and coordinated cellular responses mounted by all three cell types in tandem in modulating viraemic and aviraemic phases. This study highlights that induction of HIV-1-specific helper cells might be important in immunotherapeutic interventions and HIV vaccine development. FundingWe acknowledge the support of a National Health and Medical Research Council Development grant to NKS.

Comparison of intradermal and intramuscular delivery followed by in vivo electroporation of SIV Env DNA in macaques

A panel of SIVmac251 transmitted Env sequences were tested for expression, function and immunogenicity in mice and macaques. The immunogenicity of a DNA vaccine cocktail expressing SIVmac239 and three transmitted SIVmac251 Env sequences was evaluated upon intradermal or intramuscular injection followed by in vivo electroporation in macaques using sequential vaccination of gp160, gp120 and gp140 expressing DNAs. Both intradermal and intramuscular vaccination regimens using the gp160 expression plasmids induced robust humoral immune responses, which further improved using the gp120 expressing DNAs. The responses showed durability of binding and neutralizing antibody titers and high avidity for > 1 y. The intradermal DNA delivery regimen induced higher cross-reactive responses able to neutralize the heterologous tier 1B-like SIVsmE660_CG7V. Analysis of cellular immune responses showed induction of Env-specific memory responses and cytotoxic granzyme B+ T cells in both vaccine groups, although the magnitude of the responses were ~10x higher in the intramuscular/electroporation group. The cellular responses induced by both regimens were long lasting and could be detected ~1 y after the last vaccination. These data show that both DNA delivery methods are able to induce robust and durable immune responses in macaques.

Antigen-Specific IL-2 Secretion Correlates with NK Cell Responses after Immunization of Tanzanian Children with the RTS,S/AS01 Malaria Vaccine

RTS,S/AS01, a vaccine targeting pre-erythrocytic stages of Plasmodium falciparum, is undergoing clinical trials. We report an analysis of cellular immune response to component Ags of RTS,S-hepatitis B surface Ag (HBs) and P. falciparum circumsporozoite (CS) protein-among Tanzanian children in a phase IIb RTS,S/AS01(E) trial. RTS,S/AS01 (E) vaccinees make stronger T cell IFN-γ, CD69, and CD25 responses to HBs peptides than do controls, indicating that RTS,S boosts pre-existing HBs responses. T cell CD69 and CD25 responses to CS and CS-specific secreted IL-2 were augmented by RTS,S vaccination. Importantly, more than 50% of peptide-induced IFN-γ(+) lymphocytes were NK cells, and the magnitude of the NK cell CD69 response to HBs peptides correlated with secreted IL-2 concentration. CD69 and CD25 expression and IL-2 secretion may represent sensitive markers of RTS,S-induced, CS-specific T cells. The potential for T cell-derived IL-2 to augment NK cell activation in RTS,S-vaccinated individuals, and the relevance of this for protection, needs to be explored further.

Vaccination with Liposomal Leishmanial Antigens Adjuvanted with Monophosphoryl Lipid–Trehalose Dicorynomycolate (MPL-TDM) Confers Long-Term Protection against Visceral Leishmaniasis through a Human Administrable Route

The development of a long-term protective subunit vaccine against visceral leishmaniasis depends on antigens and adjuvants that can induce an appropriate immune response. The immunization of leishmanial antigens alone shows limited efficacy in the absence of an appropriate adjuvant. Earlier we demonstrated sustained protection against Leishmania donovani with leishmanial antigens entrapped in cationic liposomes through an intraperitoneal route. However, this route is not applicable for human administration. Herein, we therefore evaluated the immune response and protection induced by liposomal soluble leishmanial antigen (SLA) formulated with monophosphoryl lipid-trehalose dicorynomycolate (MPL-TDM) through a subcutaneous route. Subcutaneous immunization of BALB/c mice with SLA entrapped in liposomes or with MPL-TDM elicited partial protection against experimental visceral leishmaniasis. In contrast, liposomal SLA adjuvanted with MPL-TDM induced significantly higher levels of protection in liver and spleen in BALB/c mice challenged 10 days post-vaccination. Protection conferred by this formulation was sustained up to 12 weeks of immunization, and infection was controlled for at least 4 months of the challenge, similar to liposomal SLA immunization administered intraperitoneally. An analysis of cellular immune responses of liposomal SLA + MPL-TDM immunized mice demonstrated the induction of IFN-γ and IgG2a antibody production not only 10 days or 12 weeks post-vaccination but also 4 months after the challenge infection and a down regulation of IL-4 production after infection. Moreover, long-term immunity elicited by this formulation was associated with IFN-γ production also by CD8⁺ T cells. Taken together, our results suggest that liposomal SLA + MPL-TDM represent a good vaccine formulation for the induction of durable protection against L. donovani through a human administrable route.