T cell-, interleukin-12-, and gamma interferon-driven viral clearance in measles virus-infected brain tissue

Genetic studies with immunocompetent mice show the importance of both T cells and gamma interferon (IFN-γ) for survival of a measles virus (MV) challenge; however, the direct role of T cells and IFN-γ within the MV-infected brain has not been addressed. Organotypic brain explants represent a successful ex vivo system to define central nervous system (CNS)-specific mechanisms of leukocyte migration, activation, and MV clearance. Within the heterogeneous, brain-derived, primed leukocyte population which reduced MV RNA levels in brain explants by 60%, CD3 T cells are the active antiviral cells, as purified CD3-positive cells are highly antiviral and CD3-negative leukocytes are unable to reduce the viral load. Neutralization of CCL5 and CXCL10 decreases leukocyte migration to areas of infection by 70%. However, despite chemokines directing the migration of T cells to infected neurons, chemokine neutralization revealed that migration is not required for viral clearance, suggesting a cytokine-mediated antiviral mechanism. In accordance with our hypothesis, the ability of leukocytes to clear the virus is abrogated when explants are treated with anti-IFN-γ neutralizing antibodies. IFN-γ applied to infected slices in the absence of primed leukocytes reduces the viral load by more than 80%; therefore, in brain tissue, IFN-γ is both necessary and sufficient to clear MV. Secretion of IFN-γ is stimulated by interleukin-12 (IL-12) in the brain, as neutralization of IL-12 results in loss of antiviral activity and stimulation of leukocytes with IL-12/IL-18 enhances their immune effector function of viral clearance. MV-primed leukocytes can reduce both West Nile and mouse hepatitis viral RNAs, indicating that cytokine-mediated viral clearance occurs in an antigen-independent manner. The IFN-γ signal is transduced within the brain explant by the Jak/STAT signaling pathway, as inhibition of Jak kinases results in a loss of antiviral activity driven by either brain-derived leukocytes or recombinant IFN-γ. These results reveal that primed T cells directly act to clear MV infection of the brain by using a noncytolytic IL-12- and IFN-γ-dependent mechanism in the CNS and that this mechanism relies upon Jak/STAT signaling.     

Cancer targeting gene-viro-therapy specific for liver cancer by α-fetoprotein-controlled oncolytic adenovirus expression of SOCS3 and IL-24

The combination of gene therapy and virotherapy for cancer treatment has received close attention and has become a trend in the field of cancer biotherapy. A strategy called 'Cancer Targeting Gene-Viro-Therapy' (CTGVT) or 'Gene Armed Oncolytic Viral Therapy' (GAOVT) has been proposed, in which an antitumor gene is inserted into an oncolytic viral vector. In our previous study, a dual-regulated oncolytic adenovirus with enhanced safety for normal cells and strict liver cancer-targeting ability, designated Ad?enAFP?E1A?E1B (Δ55) (briefly Ad?enAFP?D55), was successfully constructed. In the current work, interleukin-24 (IL-24) and suppressor of cytokine signaling 3 (SOCS3) genes were packaged into Ad?enAFP?D55. The new constructs, Ad?enAFP?D55-(IL-24) and Ad?enAFP?D55-(SOCS3), showed improved tumoricidal activity in hepatoma cell lines compared with the oncolytic viral vector Ad?enAFP?D55. The co-administration of Ad?enAFP?D55-(IL-24) and Ad?enAFP?D55-(SOCS3) showed much better antitumor effect than Ad?enAFP?D55-(IL-24) or Ad?enAFP?D55-(SOCS3) alone both in vitro and in a nude mouse xenograft model. Moreover, our results also showed that blockade of the Jak/Stat3 pathway by Ad?enAFP?D55-(SOCS3) infection in HuH-7 cells could down-regulate some anti-apoptosis proteins, such as XIAP, Bcl-xL, and survivin, which might sensitize the cells to Ad?enAFP?D55-(IL-24)-induced apoptosis. These results indicate that co-administration of Ad?enAFP?D55-(IL-24) and Ad?enAFP?D55-(SOCS3) may serve as a candidate therapeutic approach for the treatment of liver cancer.     

Oncolytic Properties of a Mumps Virus Vaccine Strain in Human Melanoma Cell Lines

The oncolytic potential of the attenuated mumps virus (MV) vaccine strain Leningrad-3 (L-3) was evaluated in a panel of four human metastatic melanoma cell lines. The lines were shown to be susceptible and permissive to MV infection. Efficient MV replication led to death of melanoma cells, but the effect differed among the cell lines. Possible mechanisms mediating the selectivity of MV L-3 towards the cell lines were explored. Replicative and oncolytic activity of MV was found to depend on the expression pattern of type I interferon genes. None of the melanoma cell lines showed induction of expression of the total spectrum of genes required to inhibit virus replication. Based on the results, MV L-3 was assumed to be a promising oncolytic agent for human melanoma cells.     

Small molecules baicalein and cinnamaldehyde are potentiators of measles virus-induced breast cancer oncolysis

BackgroundAlthough the breast cancer mortality has slowed down from 2008 to 2017, breast cancer incidence rate continues to rise and thus, new and/or improved treatments are highly needed. Among them, oncolytic virotherapy which has the ability of facilitating the antitumor adaptive immunity, appears as a promising anticancer therapy. Oncolytic measles virus (MV) is particularly suitable for targeting breast cancer due to the upregulation of MV's receptor nectin-4. Nonetheless, with limited clinical success currently, ways of boosting MV-induced breast cancer oncolysis are therefore necessary. Oncolytic virotherapy alone and combined with chemotherapeutic drugs are two strategic areas with intensive development for the search of anticancer drugs. Considering that baicalein (BAI) and cinnamaldehyde (CIN) have demonstrated antitumor properties against multiple cancers including breast cancer, they could be good partners for MV-based oncolytic virotherapy.PurposeTo assess the in vitro effect of BAI and CIN with MV and assess their combination effects.MethodsWe examined the combinatorial cytotoxic effect of oncolytic MV and BAI or CIN on MCF-7 breast cancer cells. Potential anti-MV activities of the phytochemicals were first investigated in vitro to determine the optimal combination model. Synergism of MV and BAI or CIN was then evaluated in vitro by calculating the combination indices. Finally, cell cycle analysis and apoptosis assays were performed to confirm the mechanism of synergism.ResultsOverall, the viral sensitization combination modality using oncolytic MV to first infect MCF-7 breast cancer cells followed by drug treatment with BAI or CIN was found to produce significantly enhanced tumor killing. Further mechanistic studies showed that the combinations ‘MV-BAI’ and ‘MV-CIN’ display synergistic anti-breast cancer effect, mediated by elevated apoptosis.ConclusionWe demonstrated, for the first time, effective combination of oncolytic MV with BAI or CIN that could be further explored and potentially developed into novel therapeutic strategies targeting nectin-4-marked breast cancer cells.     

Ablation of Nectin4 Binding Compromises CD46 Usage by a Hybrid Vesicular Stomatitis Virus/Measles Virus

Measles virus (MV) immunosuppression is due to infection of SLAM-positive immune cells, whereas respiratory shedding and virus transmission are due to infection of nectin4-positive airway epithelial cells. The vaccine lineage MV strain Edmonston (MV-Edm) acquired an additional tropism for CD46 which is the basis of its oncolytic specificity. VSVFH is a vesicular stomatitis virus (VSV) encoding the MV-Edm F and H entry proteins in place of G. The virus spreads faster than MV-Edm and is highly fusogenic and a potent oncolytic. To determine whether ablating nectin4 tropism from VSVFH might prevent shedding, increasing its safety profile as an oncolytic, or might have any effect on CD46 binding, we generated VSVFH viruses with H mutations that disrupt attachment to SLAM and/or nectin4. Disruption of nectin4 binding reduced release of VSVFH from the basolateral side of differentiated airway epithelia composed of Calu-3 cells. However, because nectin4 and CD46 have substantially overlapping receptor binding surfaces on H, disruption of nectin4 binding compromised CD46 binding and greatly diminished the oncolytic potency of these viruses on human cancer cells. Thus, our results support continued preclinical development of VSVFH without ablation of nectin4 binding.     

Cancer targeting gene-viro-therapy for pancreatic cancer using oncolytic adenovirus ZD55-IL-24 in immune-competent mice

Cancer targeting gene-viro-therapy (CTGVT) may prove to be an effective treatment for pancreatic cancer (PC). This study was intended to explore the anti-tumor effect of ZD55-IL-24 (oncolytic adenovirus ZD55 harboring IL-24) on PC in immune-competent mice. The expression of gene harbored by oncolytic adenovirus ZD55 in PC cells was detected by reporter-gene assays. The in vitro anti PC ability of ZD55-IL-24 was tested by MTT, crystal violet staining and apoptosis assays. The in vivo anti PC effect of ZD55-IL-24 was further observed in an immune-competent mice model by detecting anti-tumor immunity and induction of apoptosis. The expression of gene harbored by ZD55 in PC cells was significantly higher than that harbored by the replicated-deficient adenovirus, and the amount of gene expression was time-dependent and dose-dependent. Both ZD55-IL-24 and ZD55 inhibited PC cells growth, but the anti-tumor effect of ZD55-IL-24 was significantly stronger than that of ZD55, and the ability of ZD55-IL-24 in inducing PC apoptosis was significantly stronger than that of ZD55. The tumor-forming rate of group ZD55-IL-24 was the lowest, and the tumor-growing rate was also significantly lower than that of group ZD55 in immune-competent PC models. Moreover, ZD55-IL-24 mediated more anti-cancer immunity effects by induction of stronger T-lymphocytes response to PC cells, higher levels of γ-IFN and IL-6 cytokines. ZD55-IL-24-mediated CTGVT could inhibit PC growth not only by inducing oncolysis and apoptosis but enhancing the anti-cancer immune effects by inducing T cell response to PC and up-regulating γ-IFN and IL-6 cytokine in immune-competent mice. This may serve as a candidate therapeutic approach for the treatment of PC.     

Attenuated, oncolytic, but not wild-type measles virus infection has pleiotropic effects on human neutrophil function

We previously showed that neutrophils play a role in regression of human tumor xenografts in immunodeficient mice following oncolytic vaccine measles virus (MV-Vac) treatment. In this study, we sought, using normal human neutrophils, to identify potential neutrophil-mediated mechanisms for the attenuated MV-Vac induced effects seen in vivo, by comparison with those consequent on wild-type (WT-MV) infection. Both MV-Vac and WT-MV infected and replicated within neutrophils, despite lack of SLAM expression. In both cases, neutrophils survived longer ex vivo postinfection. Furthermore, MV-Vac (but not WT-MV) infection activated neutrophils and stimulated secretion of several specific antitumor cytokines (IL-8, TNF-α, MCP-1, and IFN-α) via induction of de novo RNA and protein synthesis. In addition, MV-Vac (but not WT-MV) infection caused TRAIL secretion in the absence of de novo synthesis by triggering release of prefabricated TRAIL, via a direct effect upon degranulation. The differences between the outcome of infection by MV-Vac and WT-MV were not entirely explained by differential infection and replication of the viruses within neutrophils. To our knowledge, this is the first demonstration of potential mechanisms of oncolytic activity of an attenuated MV as compared with its WT parent. Furthermore, our study suggests that neutrophils have an important role to play in the antitumor effects of oncolytic MV.     

Prostate tumor cells infected with a recombinant influenza virus expressing a truncated NS1 protein activate cytolytic CD8+ cells to recognize noninfected tumor cells

Many viral oncolytic approaches against cancer are based on the ability of specific viruses to replicate in tumors expressing components of the constitutively activated Ras/mitogen-activated protein kinase (MAPK) pathways and/or inhibited or dysregulated alpha/beta interferon (IFN-α/β) response pathways. A major issue when considering these approaches is their applicability to tumors that lack activated Ras. To identify the effector mechanisms activated by oncolytic viruses, we investigated inhibition of proliferation of the prostate cancer line LNCap by the recombinant TR-NS1 influenza A virus, a genetically attenuated influenza A/PR8/34 virus expressing a truncated nonstructural protein (NS1) of 126 amino acids. LNCap cells lack constitutively activated MAPK, extracellular signal-regulated kinase (ERK), and p38 and are resistant to death by IFN-α. Truncation of the NS1 protein of influenza viruses is known to result in viral attenuation due to a reduced ability of the NS1 to inhibit the IFN-α/β response. Infection with TR-NS1 virus rapidly activated ERK-1 more than ERK-2 in LNCap cells. Importantly, TR-NS1 virus infection transiently inhibited cell proliferation and induced apoptosis in LNCap cells. Addition of peripheral blood mononuclear cells (PBMC) and interleukin 12 (IL-12) to TR-NS1 virus-infected LNCap cells (TR-NS1-LNCap) resulted in faster elimination of TR-NS1-LNCap cells compared with LNCap cells. Moreover, TR-NS1-LNCap cells induced IFN-γ in PBMC. The levels of IFN-γ were amplified by IL-12. TR-NS1-LNCap cells also induced tumor-lytic cytotoxic T lymphocytes (CTL). These CTL lysed noninfected LNCap cells in a CD8-dependent manner. Activation of cellular immunity to tumor cells by viruses is an intriguing effector pathway, which should be especially significant for elimination of human tumors that lack activated Ras.     

Oncolytic measles viruses produced at different scales under serum‐free conditions

Measles virus (MV) with attenuated pathogenicity has potential as oncolytic agent. However, the clinical translation of this therapy concept has one major hurdle: the production of sufficient amounts of infectious oncolytic MV particles. The current study describes oncolytic MV production in Vero cells grown on microcarrier using serum‐free medium. The impact of the number of harvests, cell concentration at infection (CCI), multiplicity of infection (MOI), and temperature on MV production was determined in different production scales/systems (static T‐flasks, dynamic spinner, and bioreactor system) and modes (batch, repeated‐batch, and perfusion). Cell growth, metabolic, and production kinetics were analyzed. It was found that the number of harvests had the strongest positive impact on MV yield in each production scale, and that high temperatures affected MV yield adversely. Moderate MV titers were produced in T‐ and spinner flasks at 37°C (～10⁷ TCID₅₀ mL^?1, where TCID₅₀ is tissue culture infective doses 50%), but stirred tank reactor (STR) MV production at 37°C yielded up to 10 000‐fold lower MV titers. In contrast, at lower temperatures (32°C, 27°C), 1.4 × 10⁷ TCID₅₀ mL^?1 were achieved in the STR. Variations in MOI and CCI had almost no influence on MV production yield. The current study improves oncolytic MV production process understanding and identifies process bottlenecks for large‐scale production.     

Measles virus induces abnormal differentiation of CD40 ligand-activated human dendritic cells

Measles virus (MV) infection induces a profound immunosuppression responsible for a high rate of mortality in malnourished children. MV can encounter human dendritic cells (DCs) in the respiratory mucosa or in the secondary lymphoid organs. The purpose of this study was to investigate the consequences of DC infection by MV, particularly concerning their maturation and their ability to generate CD8+ T cell proliferation. We first show that MV-infected Langerhans cells or monocyte-derived DCs undergo a maturation process similarly to the one induced by TNF-alpha or LPS, respectively. CD40 ligand (CD40L) expressed on activated T cells is shown to induce terminal differentiation of DCs into mature effector DCs. In contrast, the CD40L-dependent maturation of DCs is inhibited by MV infection, as demonstrated by CD25, CD69, CD71, CD40, CD80, CD86, and CD83 expression down-regulation. Moreover, the CD40L-induced cytokine pattern in DCs is modified by MV infection with inhibition of IL-12 and IL-1alpha/beta and induction of IL-10 mRNAs synthesis. Using peripheral blood lymphocytes from CD40L-deficient patients, we demonstrate that MV infection of DCs prevents the CD40L-dependent CD8+ T cell proliferation. In such DC-PBL cocultures, inhibition of CD80 and CD86 expression on DCs was shown to require both MV replication and CD40 triggering. Finally, for the first time, MV was shown to inhibit tyrosine-phosphorylation level induced by CD40 activation in DCs. Our data demonstrate that MV replication modifies CD40 signaling in DCs, thus leading to impaired maturation. This phenomenon could play a pivotal role in MV-induced immunosuppression.     

The role of cell signaling in poxvirus tropism: the case of the M-T5 host range protein of myxoma virus

Poxviruses demonstrate strict species specificity in vivo that range from narrow to broad, however the fundamental factors that mediate the basis of poxvirus tropism remain poorly understood. It is generally believed that most, if not all, poxviruses can efficiently bind and enter a wide range of mammalian cells and all of the known host anti-viral pathways that block viral replication in nonpremissive cells operate downstream of virus entry. A productive poxvirus infection is heavily dependent upon the production of a vast array of host modulatory products that specifically target and manipulate both extracellular immune response pathways of the host, as well as intracellular signal transduction pathways of the individually infected cells. The unique pathogenesis and host tropism of specific poxviruses can be attributed to the broad diversity of host modulatory proteins they express. Myxoma virus (MV) is a rabbit-specific poxviruses that encodes multiple host range factors, including an ankyrin-repeat protein M-T5, which functions to regulate tropism of MV for rabbit lymphocytes and some human cancer cells. At the molecular level, M-T5 binds and alters at least two distinct cellular proteins: Akt and cullin-1. The direct interaction between M-T5 and Akt was shown to be a key restriction determinant for MV tropism in a spectrum of human cancer cells making MV an excellent oncolytic candidate. Thus, the intricate relationship between viral encoded proteins and components of the host cell signaling networks can have profound impact on poxvirus tropism. The lessons we continue to learn from poxvirus host range factors like M-T5 will provide further insights into the factors that regulate poxvirus tropism and the mechanisms by which poxviruses micromanipulate the signaling pathways of the infected cell.     

Hemagglutinin protein of wild-type measles virus activates toll-like receptor 2 signaling

Pattern recognition via Toll-like receptors (TLR) by antigen-presenting cells is an important element of innate immunity. We report that wild-type measles virus but not vaccine strains activate cells via both human and murine TLR2, and this is a property of the hemagglutinin (H) protein. The ability to activate cells via TLR2 by wild-type MV H protein is abolished by mutation of a single amino acid, asparagine at position 481 to tyrosine, as is found in attenuated strains, which is important for interaction with CD46, the receptor for these strains. TLR2 activation by MV wild-type H protein stimulates induction of proinflammatory cytokines such as interleukin-6 (IL-6) in human monocytic cells and surface expression of CD150, the receptor for all MV strains. Confirming the specificity of this interaction, wild-type H protein did not induce IL-6 release in macrophages from TLR2-/- mice. Thus, the unique property of MV wild-type strains to activate TLR2-dependent signals might essentially contribute not only to immune activation but also to viral spread and pathogenicity by upregulating the MV receptor on monocytes.     

Screening different host cell lines for the dynamic production of measles virus

Measles virus (MV) has a natural affinity for cancer cells and oncolytic MV preparations have therefore been investigated in several clinical trials as a potential treatment for cancer. The main bottleneck in the administration of oncolytic MV to cancer patients is the production process, because very large doses of virus particles are required for each treatment. Here, we investigated the productivity of different host cells and found that a high infection efficiency did not necessarily result in high virus yields because virus release is also dependent on the host cell. As well as producing large numbers of active MV particles, host cells must perform well in dynamic cultivation systems. In screening experiments, the highest productivity was achieved by Vero and BJAB cells, but only the Vero cells maintained their high virus productivity when transferred to a stirred tank reactor. We used dielectric spectroscopy as an online monitoring system to control the infection and harvest times, which are known to be critical process parameters. The precise control of these parameters allowed us to achieve higher virus titers with Vero cells in a stirred tank reactor than in a static cultivation system based on T‐flasks, with maximum titers of up to 10¹¹ TCID₅₀ ml^?1. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:989–997, 2017     

Oncolytic effects of a novel influenza A virus expressing interleukin-15 from the NS reading frame

Oncolytic influenza A viruses with deleted NS1 gene (delNS1) replicate selectively in tumour cells with defective interferon response and/or activated Ras/Raf/MEK/ERK signalling pathway. To develop a delNS1 virus with specific immunostimulatory properties, we used an optimised technology to insert the interleukin-15 (IL-15) coding sequence into the viral NS gene segment (delNS1-IL-15). DelNS1 and delNS1-IL-15 exerted similar oncolytic effects. Both viruses replicated and caused caspase-dependent apoptosis in interferon-defective melanoma cells. Virus replication was required for their oncolytic activity. Cisplatin enhanced the oncolytic activity of delNS1 viruses. The cytotoxic drug increased delNS1 replication and delNS1-induced caspase-dependent apoptosis. Interference with MEK/ERK signalling by RNAi-mediated depletion or the MEK inhibitor U0126 did not affect the oncolytic effects of the delNS1 viruses. In oncolysis sensitive melanoma cells, delNS1-IL-15 (but not delNS1) infection resulted in the production of IL-15 levels ranging from 70 to 1140 pg/mL in the cell culture supernatants. The supernatants of delNS1-IL-15-infected (but not of delNS1-infected) melanoma cells induced primary human natural killer cell-mediated lysis of non-infected tumour cells. In conclusion, we constructed a novel oncolytic influenza virus that combines the oncolytic activity of delNS1 viruses with immunostimulatory properties through production of functional IL-15. Moreover, we showed that the oncolytic activity of delNS1 viruses can be enhanced in combination with cytotoxic anti-cancer drugs.     

Interleukin 12: still a promising candidate for tumor immunotherapy?

Interleukin 12 (IL-12) seemed to represent the ideal candidate for tumor immunotherapy, due to its ability to activate both innate (NK cells) and adaptive (cytotoxic T lymphocytes) immunities. However, despite encouraging results in animal models, very modest antitumor effects of IL-12 in early clinical trials, often accompanied by unacceptable levels of adverse events, markedly dampened  hopes of the successful use of this cytokine in cancer patients. Recently, several clinical studies have been initiated in which IL-12 is applied as an adjuvant in cancer vaccines, in gene therapy including locoregional injections of IL-12 plasmid and in the form of tumor-targeting immunocytokines (IL-12 fused to monoclonal antibodies). The near future will show whether this renewed interest in the use of IL-12 in oncology will result in meaningful therapeutic effects in a select group of cancer patients.     

Nectin 4 is the epithelial cell receptor for measles virus

Measles virus (MV) causes acute respiratory disease, infects lymphocytes and multiple organs, and produces immune suppression leading to secondary infections. In rare instances it can also cause persistent infections in the brain and central nervous system. Vaccine and laboratory-adapted strains of MV use CD46 as a receptor, whereas wild-type strains of MV (wtMV) cannot. Both vaccine and wtMV strains infect lymphocytes, monocytes, and dendritic cells (DCs) using the signaling lymphocyte activation molecule (CD150/SLAM). In addition, MV can infect the airway epithelial cells of the host. Nectin 4 (PVRL4) was recently identified as the epithelial cell receptor for MV. Coupled with recent observations made in MV-infected macaques, this discovery has led to a new paradigm for how the virus accesses the respiratory tract and exits the host. Nectin 4 is also a tumor cell marker which is highly expressed on the apical surface of many adenocarcinoma cell lines, making it a potential target for MV oncolytic therapy.     

Cytokine Imbalance after Measles Virus Infection Has No Correlation with Immune Suppression

Measles virus infection leads to immune suppression. A potential mechanism is the reduction of interleukin 12 (IL-12) secretion during acute measles, resulting in a TH2 response. Studies in humans have reported conflicting results, detecting either a TH2 or a TH1 response. We have investigated the correlation between a TH2 response and immune suppression in specific-pathogen-free inbred cotton rats which were infected with measles vaccine and wild-type viruses. After infection of bone marrow-derived macrophages with wild-type virus, IL-12 secretion was reduced in contrast to the level for vaccine virus infection. In bronchoalveolar lavage cells, IL-12 secretion was suppressed after infection with both wild-type and vaccine virus on days 2, 4, and 6 and was detectable on days 8 and 10. After stimulation of mediastinal lymph node and spleen cells with UV-inactivated measles virus at various time points after infection, gamma interferon but no IL-4 was found. After stimulation with phorbol myristate acetate-ionomycin, high gamma interferon and low IL-4 levels were detected. To investigate whether the secretion of IL-4 contributes to immune suppression, a recombinant vaccine virus was created which secretes cotton rat IL-4. After infection with this recombinant virus, IL-4 secretion was enhanced. However, neither inhibition of concanavalin A-stimulated spleen cells nor keyhole limpet hemocyanin-specific proliferation of spleen cells was altered after infection with the recombinant virus in comparison to the levels with the parental virus. Our data indicate that measles virus infection leads to a decrease in IL-12 secretion and an increase in IL-4 secretion, but this does not seem to correlate with immune suppression.Acute measles is caused by infection with measles virus (MV) and is associated with high morbidity and mortality. The main reason for these is thought to be immune suppression due to MV infection. Studies addressing the mechanism of immune suppression have found evidence for a number of possible mechanisms. These include unidentified soluble mediators (10, 39, 40), interference with the type I interferon (IFN) system (for a review, see reference 9), apoptosis (8), impaired lymphoproliferation (23, 35), interleukin 12 (IL-12) downregulation (1, 18), and impaired dendritic cell (DC) function (36). Some of the mechanisms may operate in conjunction with each other; e.g., it is possible that the downregulation of IL-12 might lead to the development of a T-helper 2 (TH2) response that results in impaired lymphoproliferation. The evidence for this line of argument is that human macrophages, after stimulation through Toll-like receptor 4 (TLR-4), secrete less IL-12 when infected with MV (18). In MV-infected humans, the percentage of IL-12-expressing macrophages is reduced (1), and in Rhesus macaques, lowered serum levels of IL-12 have been found during MV infection (31). In macaques, this correlates with an increase of eosinophils in peripheral blood, indicative of increased secretion of IL-5 (31).In serum or supernatants of peripheral blood lymphocytes (PBLs) stimulated with phorbol myristate acetate (PMA)-ionomycin from patients with acute measles, an upregulation of IFN-γ and IL-2 (12) (or IL-2-receptor [37]) was found before and during the rash, whereas after the rash, a reduction in IL-2 and an increase in IL-4 (12) or IL-10 (22) levels were observed. In contrast, other studies found normal levels of IFN-γ and IL-2 secreted by stimulated PBLs (42) and increased levels of IFN-γ after the rash secreted by PMA-ionomycin-stimulated CD8 T cells (22).Results from the analysis of cytokines secreted by lymphocytes from MV vaccinees were also conflicting. In one study, an upregulation of IL-4 and downregulation of IFN-γ were found in the supernatants of phytohemagglutinin-stimulated cells (41). In another study, increased IL-12 and IFN-γ levels were detected after stimulation with MV antigen (11). In a third study, no differences in the expression levels of IL-4, IL-5, or IL-12 were found between vaccinated and unvaccinated individuals by ex vivo hybridization for cytokine mRNA in PBLs (21). However, when PBLs from vaccinees were stimulated with MV and analyzed on a per-cell basis by limiting dilution, it was found that nearly all T cells expressed IFN-γ, either alone or in combination with IL-4 (16). In a similar cohort of vaccinees, it could demonstrated by a IL-4-receptor-blocking enzyme-linked immunosorbent assay (ELISA) that PBLs from about half of the individuals secreted IL-4 (7) (IFN-γ secretion was not addressed). This might be explained by a recent report suggesting that the secretion of IFN-γ and IL-4 after MV stimulation is linked to the haplotype of human major histocompatibility complex class I (MHC-I) molecules (HLA) (28).Based on these data, no clear correlation can be drawn between reduced levels of IL-12, the secretion of TH2-type cytokines, and the inhibition of lymphocyte proliferation. Furthermore, at least three variables seem to confound the analysis: (i) the use of samples from patients with acute measles (wild-type virus) versus vaccinees (vaccine virus), (ii) the method of stimulation of cells and cytokine detection, and (iii) the individual genetic makeups of the study participants.In this report, we have used inbred cotton rats to compare the abilities of wild-type and vaccine MVs to inhibit IL-12 secretion, and we have compared the secretion of TH1 and -2 cytokines after stimulation with MV antigen versus that with PMA-ionomycin. In addition, we tested a recombinant MV expressing cotton rat IL-4 for its ability to suppress the immune system.     

Immunogenicity of a Recombinant Measles-HIV-1 Clade B Candidate Vaccine

Live attenuated measles virus is one of the most efficient and safest vaccines available, making it an attractive candidate vector for a HIV/AIDS vaccine aimed at eliciting cell-mediated immune responses (CMI). Here we have characterized the potency of CMI responses generated in mice and non-human primates after intramuscular immunisation with a candidate recombinant measles vaccine carrying an HIV-1 insert encoding Clade B Gag, RT and Nef (MV1-F4). Eight Mauritian derived, MHC-typed cynomolgus macaques were immunised with 10⁵ TCID₅₀ of MV1-F4, four of which were boosted 28 days later with the same vaccine. F4 and measles virus (MV)-specific cytokine producing T cell responses were detected in 6 and 7 out of 8 vaccinees, respectively. Vaccinees with either M6 or recombinant MHC haplotypes demonstrated the strongest cytokine responses to F4 peptides. Polyfunctional analysis revealed a pattern of TNFα and IL-2 responses by CD4+ T cells and TNFα and IFNγ responses by CD8+ T cells to F4 peptides. HIV-specific CD4+ and CD8+ T cells expressing cytokines waned in peripheral blood lymphocytes by day 84, but CD8+ T cell responses to F4 peptides could still be detected in lymphoid tissues more than 3 months after vaccination. Anti-F4 and anti-MV antibody responses were detected in 6 and 8 out of 8 vaccinees, respectively. Titres of anti-F4 and MV antibodies were boosted in vaccinees that received a second immunisation. MV1-F4 carrying HIV-1 Clade B inserts induces robust boostable immunity in non-human primates. These results support further exploration of the MV1-F4 vector modality in vaccination strategies that may limit HIV-1 infectivity.     

Generation of MHC-nonrestricted and restricted oncolytic subsets from human bone marrow.

We analyzed the cytotoxicity and characterized the phenotype of oncolytic bone marrow (BM) lymphocyte subsets generated in vitro by interleukin-2 (IL-2) and stimulator cells (SC). Two irradiated B-lymphoblastoid cell lines (Daudi and EBV-transformed BSM) and fresh human acute myelogenous leukemia (AML) were used as SC. Stimulation with Daudi and IL-2 resulted in a substantial increase in cytotoxic activity (100- to 1000-fold) against a broad range of tumor targets, and total cellular expansion was higher compared to stimulation with IL-2 alone. The most prominent increase was observed in the CD16+ and CD56+/CD3- natural killer (NK) cell subset; however, a significant increase was also observed in CD56+/CD3+ T cells. Functional analysis of Daudi- and IL-2-generated subsets using fluorescence-activated cell sorting (FACS) revealed that most of the lytic activity was mediated by NK cells. Significant potentiation of oncolytic activity and cell growth was also seen in the cultures stimulated with BSM or fresh AML and IL-2. The highest oncolytic activity in the latter cultures was mediated primarily by CD8+, CD3+, and CD56- T cells, although NK cells also participated in cytotoxic activity. The T cell-mediated cytotoxicity was restricted by the major histocompatibility complex (MHC), since most cytotoxicity could be blocked by HLA I antibodies. Additionally, we observed that optimum stimulation of cytotoxicity required effector cell-stimulator cell contact. These data indicate that depending on the tumor used for stimulation, different lymphocyte subsets may be generated in IL-2 cultures. These different approaches may be useful in both specific and nonspecific immunotherapy.     

Functional modulation of human macrophages through CD46 (measles virus receptor): production of IL-12 p40 and nitric oxide in association with recruitment of protein-tyrosine phosphatase SHP-1 to CD46

Human CD46, formerly membrane cofactor protein, binds and inactivates complement C3b and serves as a receptor for measles virus (MV), thereby protecting cells from homologous complement and sustaining systemic measles infection. Suppression of cell-mediated immunity, including down-regulation of IL-12 production, has been reported on macrophages (Mphi) by cross-linking their CD46. The intracellular events responsible for these immune responses, however, remain unknown. In this study, we found that 6- to 8-day GM-CSF-treated peripheral blood monocytes acquired the capacity to recruit protein-tyrosine phosphatase SHP-1 to their CD46 and concomitantly were able to produce IL-12 p40 and NO. These responses were induced by stimulation with mAbs F(ab')(2) against CD46 that block MV binding or by a wild-type MV strain Kohno MV strain (KO; UV treated or untreated) that was reported to induce early phase CD46 down-regulation. Direct ligation of CD46 by these reagents, but not intracellular MV replication, was required for these cellular responses. Interestingly, the KO strain failed to replicate in the 6- to 8-day GM-CSF-cultured Mphi, while other MV strains replicated to form syncytia under the same conditions. When stimulated with the KO strain, rapid and transient dissociation of SHP-1 from CD46 was observed. These and previous results provide strong evidence that CD46 serves as a signal modulatory molecule and that the properties of ligands determine suppression or activation of an innate immune system at a specific maturation stage of human Mphi.