A single, oral dose of the TLR7 agonist JNJ-64794964 induces transcriptomic and phenotypic changes in peripheral immune cells in healthy adults.

BACKGROUND: Chronic hepatitis B (CHB) is responsible for major disease burden worldwide. However, the number of available therapies is limited; cure remains an elusive goal. JNJ-64794964 (JNJ-4964) is an oral toll-like receptor-7 (TLR7) agonist being evaluated for the treatment of CHB. Here, we investigated the capacity of JNJ-4964 to induce transcriptomic and immune cell changes in peripheral blood in healthy volunteers. METHODS: Peripheral blood was collected in the JNJ-4964 first-in-human phase 1 trial at multiple time points to assess transcriptomics and changes in frequency and phenotype of peripheral-blood mononuclear cells. Correlation of changes to JNJ-4964 exposure (Cmax) and changes in cytokine levels (C-X-C motif chemokine ligand 10 [CXCL10] and interferon alpha [IFN-α]) were evaluated. RESULTS: Fifty-nine genes, mainly interferon-stimulated genes, were up-regulated between 6 hours and 5 days after JNJ-4964 administration. JNJ-4964 increased frequencies of CD69, CD134, CD137, and/or CD253-expressing natural killer (NK) cells, indicative of NK cell activation. These changes correlated with Cmax, increase of CXCL10, and induction of IFN-α and were observed at IFN-α levels that are associated with no/acceptable flu-like adverse events. JNJ-4964 administration resulted in increased frequencies of CD86-expressing B cells, indicative of B-cell activation. These changes were predominantly observed at high IFN-α levels, which are associated with flu-like adverse events. CONCLUSIONS: JNJ-4964 administration led to changes in transcriptional profiles and immune cell activation phenotype, particularly for NK cells and B cells. Together, these changes could represent a set of biomarkers for the characterization of the immune response in CHB patients receiving TLR7 agonists.

Unusual outcome of in utero infection and subsequent postnatal super-infection with different PCV2b strains.

VC2002, isolated from postweaning multisystemic wasting syndrome (PMWS)-affected pig, is a mixture of two porcine circovirus genotype 2b (PCV2b) viruses, K2 and K39. Preliminary experiments disclosed short-term adverse effects of K39, but not K2, on porcine foetuses. These findings led to the hypothesis that infection of immuno-incompetent foetuses with K2 confers a status of immunotolerance, and postnatal super-infection with K39 triggers PMWS. To explore this hypothesis, nine 55-day-old foetuses were inoculated in utero (three with K2-10(4.3)TCID50, three with K39-10(4.3)TCID50 and three with medium), and foeto-pathogenicity examined. At 21 days post-inoculation (dpi), K2 did not induce pathology, whereas pathological effects of K39 were evident. Twenty-four 45-day-old foetuses were subsequently inoculated to examine the long-term effect of K2, including six with K2-high dose-10(4.3)TCID50, six with K2-low dose-10(2.3)TCID50 and 12 mock-inoculated controls. Both doses resulted in five mummified foetuses and one live-born piglet each (69dpi). K2 was recovered from all mummies. K2 and K2-specific antibodies were not detected in serum of the two live-born piglets at birth, indicating full control of K2 infection. The K2-low dose-infected piglet was immunostimulated at day 2, but not the K2-high dose-infected piglet. Both non-stimulated and stimulated K2-infected piglets were super-inoculated with K39 at day 6 or 8 (taken as 0 days post super-inoculation). Low viral replication was observed in the non-stimulated K2-K39 piglet (up to 10(3.3)TCID50/g; identified as K39). In contrast, viral replication was extremely high in the stimulated K2-K39 piglet (up to 10(5.6)TCID50/g) and identified as K2, indicating that K2 infection is controlled during foetal life, but emerges after birth upon immunostimulation. However, none of the piglets showed any signs of PMWS.

Porcine circovirus 2 uses heparan sulfate and chondroitin sulfate B glycosaminoglycans as receptors for its attachment to host cells.

Monocyte/macrophage lineage cells are target cells in vivo for porcine circovirus 2 (PCV2) replication. The porcine monocytic cell line 3D4/31 supports PCV2 replication in vitro, and attachment and internalization kinetics of PCV2 have been established in these cells. However, PCV2 receptors remain unknown. Glycosaminoglycans (GAG) are used by several viruses as receptors. The present study examined the role of GAG in attachment and infection of PCV2. Heparin, heparan sulfate (HS), chondroitin sulfate B (CS-B), but not CS-A, and keratan sulfate reduced PCV2 infection when these GAG were incubated with PCV2 prior to and during inoculation of 3D4/31 cells. Enzymatic removal of HS and CS-B prior to PCV2 inoculation of 3D4/31 cells significantly reduced PCV2 infection. Similarly, when PCV2 virus-like particles (VLP) were allowed to bind onto 3D4/31 cells in the presence of heparin and CS-B, attachment was strongly reduced. Titration of field isolates and low- and high-passage laboratory strains of PCV2 in the presence of heparin significantly reduced PCV2 titers, showing that the capacity of PCV2 to bind GAG was not acquired during in vitro cultivation but is an intrinsic feature of wild-type virus. When Chinese hamster ovary (CHO) cells were inoculated with PCV2, relative percentages of PCV2-infected cells were 27% +/- 8% for HS-deficient and 12% +/- 10% for GAG-deficient cells compared to wild-type cells (100%). Furthermore, it was shown using heparin-Sepharose chromatography that both PCV2 and PCV2 VLP directly interacted with heparin. Together, these results show that HS and CS-B are attachment receptors for PCV2.

Correlation between the presence of neutralizing antibodies against porcine circovirus 2 (PCV2) and protection against replication of the virus and development of PCV2-associated disease.

BACKGROUND: In a previous study, it was demonstrated that high replication of Porcine circovirus 2 (PCV2) in a gnotobiotic pig was correlated with the absence of PCV2-neutralizing antibodies. The aim of the present study was to investigate if this correlation could also be found in SPF pigs in which PMWS was experimentally reproduced and in naturally PMWS-affected pigs. RESULTS: When looking at the total anti-PCV2 antibody titres, PMWS-affected and healthy animals seroconverted at the same time point, and titres in PMWS-affected animals were only slightly lower compared to those in healthy animals. In healthy animals, the evolution of PCV2-neutralizing antibodies coincided with that of total antibodies. In PMWS-affected animals, neutralizing antibodies could either not be found (sera from field studies) or were detected in low titres between 7 and 14 DPI only (sera from experimentally inoculated SPF pigs). Differences were also found in the evolution of specific antibody isotypes titres against PCV2. In healthy pigs, IgM antibodies persisted until the end of the study, whereas in PMWS-affected pigs they quickly decreased or remained present at low titres. The mean titres of other antibody isotypes (IgG1, IgG2 and IgA), were slightly lower in PMWS-affected pigs compared to their healthy group mates at the end of each study. CONCLUSION: This study describes important differences in the development of the humoral immune response between pigs that get subclinically infected with PCV2 and pigs that experience a high level of PCV2-replication which in 3 of 4 experiments led to the development of PMWS. These observations may contribute to a better understanding of the pathogenesis of a PCV2-infection.

Enhancement of porcine circovirus 2 replication in porcine cell lines by IFN-gamma before and after treatment and by IFN-alpha after treatment.

Stimulation of the porcine immune system causes increased replication of porcine circovirus 2 (PCV2) in vivo. In the present study, we investigated whether various cytokines (interleukin-1 [IL-1], IL-6, IL-10, tumor necrosis factor-alpha [TNF-alpha], interferon-alpha [IFN-alpha], and IFN-gamma) are able to influence PCV2 infection in vitro. No changes were observed in IL-1, IL-6, TNF-alpha, or IL-10-treated cells. However, it was demonstrated that IFN- alpha and IFN-gamma influenced PCV2 infection in porcine kidney cells (PK-15) and porcine monocytic cells (3D4/31). IFN-gamma added to the culture medium before, during, or after inoculation increased the number of PCV2 antigen- positive cells, respectively, by 418%, 171%, and 691% in PK-15 cells and by 706%, 114%, and 423% in 3D4/31 cells. IFN-alpha pretreatment decreased the number of infected PK-15 cells. When it was added after inoculation, IFN-alpha enhanced PCV2 infection by 529% in PK-15 cells and by 308% in 3D4/31 cells. The effect of both IFNs on PCV2 infection was dose dependent and could be blocked with IFN-alpha or IFN-gamma neutralizing antibodies. Leukocyte-derived porcine IFN-gamma induced a similar effect on PCV2 infection. Treatment of PK- 15 cultures with IFN-gamma caused a 20 times higher production of progeny virus. Confocal microscopy studies showed that the enhancing effect of IFN-gamma on PCV2 infection was achieved by increased internalization of PCV2 virionlike particles (VLPs). Binding of the VLPs to the cell or expression kinetics of PCV2 proteins in infected cells were not altered by IFN-gamma treatment. To our knowledge, this study reports the first enhancement of a viral infection by treatment with type I or type II IFNs.

Characteristics of porcine circovirus-2 replication in lymphoid organs of pigs inoculated in late gestation or postnatally and possible relation to clinical and pathological outcome of infection.

In this study, the characteristics of porcine circovirus-2 (PCV2) replication (infectious virus titrations, distribution, and immunophenotyping of infected cells) in lymphoid organs were examined and related to the development of clinical signs and histological lesions in 26 piglets that had been inoculated with PCV2 either in utero or at 1 day of age. Piglets inoculated in utero at 92 or 104 gestational days (n = 12) were collected by Caesarean section at term and either sacrificed immediately or kept in isolators and allowed to live postnatally until 35 days postinoculation (PI). Caesarean-derived piglets inoculated at 1 day of age (n = 14) were sacrificed at 10, 21, 35, 42, and 49 days PI. Spleen and lymph nodes were collected for virologic and histopathological examinations. Clinical signs were not observed in any of the piglets. High virus titers (10(4.5-5.7) TCID50/g [TCID refers to tissue culture infectious dose]) were detected in 6 of the 26 piglets. Three of these 6 piglets were euthanized at 10 days PI, and infected cells of the monocyte-macrophage lineage (SWC3+, CD14+, and sialoadhesin [Sa]+ cells) and infected cells bearing lymphocyte markers (CD4+, CD8+, and immunoglobulin M+ cells) were identified by double-immunofluorescence labeling on serial cryostat sections. The other 3 piglets were euthanized at 21 and 35 days PI, and the majority of infected cells were SWC3+, CD14+, and Sa-. The absence of Sa in these infected cells, together with their localization in lymphocyte-dependent regions, suggests that they were infiltrating monocytic cells. Sialoadhesin is highly expressed in differentiated macrophages and not in peripheral blood mononuclear cells. In all 6 piglets with high virus titers, lymphocyte depletion and infiltration of monocytic cells were observed. In the remaining 20 piglets with virus titers less than 10(4.5) TCID50/g, the majority of infected cells were SWC3+, CD14+, and Sa+. In conclusion, it can be stated that high PCV2 titers in lymphoid organs may lead to the development of histological lesions similar to those observed in pigs with postweaning multisystemic wasting syndrome without causing disease. Furthermore, in lymphoid organs with high virus titers, infection occurs mainly in infiltrating monocytic cells and to a limited extent in cells bearing lymphocyte markers.

Change of porcine circovirus 2 target cells in pigs during development from fetal to early postnatal life.

Change of porcine circovirus 2 (PCV2) target cells during development from fetal to postnatal life in pigs was examined. PCV2 inoculation was performed in fetuses in utero at either 57, 75 or 92 gestational days and in piglets at 1 day of age. Twenty-one days after virus inoculation, PCV2-infected cells in the heart, lungs, liver, spleen and inguinal lymph nodes were localized and immuno-phenotyped by double-immunofluorescence labeling using different cell markers and PCV2-antibodies. During fetal life, viral antigens were detected in cardiomyocytes, hepatocytes and macrophages and infected cell numbers decreased with increasing fetal age at inoculation. The heart contained the highest number of infected cells and cardiomyocytes were the main target cell. Postnatally, macrophages were the only target cell type in different organs and infected cell numbers were similar to those of fetuses inoculated at 92 days of gestation. One piglet showed exceptionally high number of infected cells in different organs with values 13-513-fold higher compared to littermates. In this piglet, the majority of infected cells in lymphoid tissues could not be typed. This study reveals that PCV2 target cells change from cardiomyocytes, hepatocytes and macrophages during fetal life to only macrophages postnatally.