Primary Human B Cells at Different Differentiation and Maturation Stages Exhibit Distinct Susceptibilities to Vaccinia Virus Binding and Infection

Nicole Shepherd; Jie Lan; Wei Li; Sushmita Rane; Qigui Yu

doi:10.1128/JVI.00973-19

. 2019 Sep 12;93(19):e00973-19. doi: 10.1128/JVI.00973-19

Primary Human B Cells at Different Differentiation and Maturation Stages Exhibit Distinct Susceptibilities to Vaccinia Virus Binding and Infection

Nicole Shepherd ^a, Jie Lan ^a, Wei Li ^a, Sushmita Rane ^a, Qigui Yu ^a,^✉

Editor: Rozanne M Sandri-Goldin^b

PMCID: PMC6744248 PMID: 31292245

Our results provide critical information to the field of poxvirus binding and infection tropism. We demonstrate that VACV preferentially infects memory B cells that play an important role in a rapid and vigorous antibody-mediated immune response upon reinfection by a pathogen. Additionally, this work highlights the potential of B cells as natural cellular models to identify VACV receptors or dissect the molecular mechanisms underlying key steps of the VACV life cycle, such as binding, penetration, entry, and replication in primary human cells. The understanding of VACV biology in human primary cells is essential for the development of a safe and effective live-virus vector for oncolytic virus therapy and vaccines against smallpox, other pathogens, and cancer.

KEYWORDS: B cells, binding, early gene, infection, late gene, plasma cells, vaccinia virus

ABSTRACT

Vaccinia virus (VACV), the prototypical member of the poxvirus family, was used as a live-virus vaccine to eradicate smallpox worldwide and has recently received considerable attention because of its potential as a prominent vector for the development of vaccines against infectious diseases and as an oncolytic virus for cancer therapy. Studies have demonstrated that VACV exhibits an extremely strong bias for binding to and infection of primary human antigen-presenting cells (APCs), including monocytes, macrophages, and dendritic cells. However, very few studies have assessed the interactions of VACV with primary human B cells, a main type of professional APCs. In this study, we evaluated the susceptibility of primary human peripheral B cells at various differentiation and maturation stages to VACV binding, infection, and replication. We found that plasmablasts were resistant to VACV binding, while other B subsets, including transitional, mature naive, memory, and plasma cells, were highly susceptible to VACV binding. VACV binding preference was likely associated with differential expression of chemokine receptors, particularly CXCR5. Infection studies showed that plasmablast, plasma, transitional, and mature naive B cells were resistant to VACV infection, while memory B cells were preferentially infected. VACV infection in ex vivo B cells was abortive, which occurred at the stage of late viral gene expression. In contrast, activated B cells were permissive to productive VACV infection. Thus, primary human B cells at different differentiation stages exhibit distinct susceptibilities to VACV binding and infection, and the infections are abortive and productive in ex vivo and activated B cells, respectively.

IMPORTANCE Our results provide critical information to the field of poxvirus binding and infection tropism. We demonstrate that VACV preferentially infects memory B cells that play an important role in a rapid and vigorous antibody-mediated immune response upon reinfection by a pathogen. Additionally, this work highlights the potential of B cells as natural cellular models to identify VACV receptors or dissect the molecular mechanisms underlying key steps of the VACV life cycle, such as binding, penetration, entry, and replication in primary human cells. The understanding of VACV biology in human primary cells is essential for the development of a safe and effective live-virus vector for oncolytic virus therapy and vaccines against smallpox, other pathogens, and cancer.

INTRODUCTION

Poxviruses are a large family of DNA viruses that have recently attracted substantial attention for their potential use as bioterrorism agents (1), emerging zoonotic infections that are being increasingly reported worldwide (2 –6), clinical application for oncolytic virus therapy (7 –9), and the development of vaccine vectors against infectious pathogens and cancer (10, 11). Variola virus, the causative agent of smallpox, is the most notorious member of poxviruses that represented a great threat to humans for centuries. This virus could be used as a devastating bioweapon due to the unaccounted-for virus stocks in decommissioned laboratories and the vast majority of the population lacking vaccination with vaccinia virus (VACV), the prototypical poxvirus used as a live-virus vaccine to eradicate smallpox (12). In addition to the threat of smallpox, monkeypox, which is caused by infection with monkeypox virus, represents an emerging deadly disease in humans (13). Monkeypox can spread from animals to humans fairly easily if humans come into contact with infected animals, leading to a disfiguring smallpox-like disease with a mortality rate of approximately 10% in Africa (13). While monkeypox is endemic to Africa, an outbreak infecting 47 individuals occurred in 2003 in the United States (5, 14). Several other poxviruses, including buffalopox, cowpox, tanapox, and Cantagalo virus, are also emerging in South America, Africa, Europe, India, and the United States (6, 15 –18). Thus, human zoonotic poxvirus infections are increasingly encountered outside their usual geographical ranges. However, besides these threats to life and health of humans, several poxviruses have been intensively studied as live-virus vectors for oncolytic virus therapy and vaccine development to control infectious diseases in animals and humans (11, 19 –30). Poxviruses such as VACV have a safe profile in humans (31), a broad infection spectrum of tumor cells (32), a rapid cytolytic replication cycle (33), a strict cytoplasmic life cycle with no DNA integration (31, 34), and a large DNA genome to allow stable incorporations of large transgenes (35). These unique biological features make them ideal candidates as oncolytic agents for cancer therapy and vaccine vectors against infectious diseases. A canarypox-vectored HIV vaccine, known as RV144, demonstrated moderate success after a 6-year clinical trial on more than 16,000 volunteers in Thailand (11). To date, this is the only HIV vaccine showing evidence of protection against HIV infection, suggesting that poxviruses are promising vectors for development of vaccines against pathogens such as HIV and cancer.

Poxviruses, including VACV, demonstrate an extremely strong preference for binding to and infection of primary human antigen-presenting cells (APCs), including monocytes, macrophages, and dendritic cells (DCs) (36 –39). However, very few studies have evaluated VACV binding to and infection of primary human B cells that are a main type of APCs in addition to producing antibodies. Clinical studies and animal experiments have demonstrated that B cells are involved in poxvirus infection and antiviral memory immune responses (4, 36 –38). In VACV zoonotic outbreaks in Brazil, individuals acutely infected with VACV exhibit decreased peripheral B cell counts and activation compared to levels for uninfected individuals, indicating that VACV targets B cells in some manner (4). Additionally, antibodies in VACV-vaccinated macaques play a major role in long-term protection against monkeypox virus infection (40), indicating that VACV-specific B cell responses are important for protection from monkeypox. Therefore, it is important to evaluate the interactions of poxviruses such as VACV with primary B cells to better understand the effect of poxvirus infection on B cell biology.

B cells comprise 5% to 15% of total lymphocytes in the human body and play an important role in the adaptive immune system (41). In addition to producing antibodies, B cells perform critical immune functions, including cytokine production, generation of immunological memory, costimulation of T cells, regulation of DC function, and antigen presentation (41, 42). Disruption of B cell development or function results in autoimmune diseases, immunodeficiency disorders, and malignancies (41, 42). B cells are a highly heterogeneous population of cells at different stages of maturation and differentiation, each with unique functional properties and cell surface phenotypes. Development of B cells begins with common lymphoid progenitors (CLPs) that progress through the early stages of B cell maturation in the bone marrow. Immature B cells exit the bone marrow through the blood to migrate to peripheral lymphoid tissues, where they mature to express IgM and IgD. Upon engagement with an antigen and costimulatory signals, mature B cells migrate to lymphoid tissue germinal centers (GCs) and subsequently exit to differentiate into either memory B cells, plasmablasts, or plasma cells to provide immediate or persistent protection (43). According to B cell maturation stages, circulating peripheral blood B cells can be classified into (i) transitional, (ii) mature naive, (iii) memory, and (iv) plasmablasts/plasma cells. Memory B cells can be further classified into nonswitched memory, IgM-only memory, and class-switched memory subsets based on their differential expression of unique Ig heavy chain of IgD or IgM isotypes (41, 42). It remains unknown how VACV interacts with circulating peripheral blood B cells at different maturation stages. In the present study, we investigated VACV binding to and infection of primary human B cells at various stages of cell maturation and differentiation in the blood. We found that plasmablasts were resistant to VACV binding, while B cells at other differentiation and maturation stages exhibited no defects in VACV binding. Plasmablast, plasma, mature naive, and transitional B cell subsets were resistant to VACV infection, while memory B cells were susceptible to VACV infection. VACV infection in ex vivo B cells was aborted at the late stage of viral gene expression.

RESULTS

VACV robustly bound to but moderately or weakly infected primary human B cells.

Studies using peripheral blood mononuclear cells (PBMCs) from healthy blood donors have demonstrated that ex vivo APCs, including monocytes, dendritic cells, and B cells, displayed robust VACV binding (39, 44), while only moderate or weak infection was seen in B cells (36, 38, 39, 44). To better understand this difference between binding and infection, we first examined if this disparity was recapitulated in isolated B cells by assessing VACV binding and infection in isolated ex vivo B cells. We found that the highly purified (purity of >97% CD19⁺) ex vivo B cells were highly susceptible to VACV binding but moderately or weakly infected by VACV (Fig. 1). These binding and infection results were in agreement with observations in PBMCs from previous studies (39, 44). Since B cells were positively isolated using the pan-B cell marker of CD19, these isolated B cells contained CD20^hi transitional and mature B cells and CD20^lo B cells such as plasmablasts and plasma cells. We next did surface staining of ex vivo B cells with a fluorochrome-conjugated antibody against human CD20 to evaluate susceptibility of CD19⁺ CD20^lo B cells and CD19⁺ CD20^hi B cells to VACV binding and infection. We observed that 58.3% ± 5.1% (n = 5) of CD19⁺ CD20^hi B cells and 22.6% ± 2.3% (n = 5) of CD19⁺ CD20^lo B cells were bound with VACV (Fig. 1A and B). Thus, VACV binding showed a strong bias toward CD19⁺ CD20^hi B cells rather than CD19⁺ CD20^lo B cells (Fig. 1A and B). In one of our previous reports, we demonstrated that VACV receptors were highly enriched in the lipid rafts of primary human monocytes/macrophages (44). To test whether VACV binding molecules are enriched in lipid rafts of ex vivo B cells, we studied colocalization of VACV binding with lipid rafts on the surface of B cells. As shown in Fig. 1C, colocalization of VACV with lipid rafts on B cells was observed, indicating that VACV receptors are strongly associated with lipid rafts in ex vivo B cells. In comparison to VACV binding, both CD19⁺ CD20^hi B cells and CD19⁺ CD20^lo B cells exhibited decreased susceptibility to VACV infection. After 12 h of infection with VV-EGFP, a recombinant VACV containing a chimeric gene that encodes the influenza virus nucleoprotein, the ovalbumin SIINFEKL peptide, and enhanced green fluorescent protein (EGFP) under the control of the P7.5 early/late promoter, 14.2% ± 3.9% (n = 3) and 10.6% ± 5.1% (n = 3) of CD19⁺ CD20^hi and CD19⁺ CD20^lo B cells, respectively, became EGFP positive (Fig. 1D and E). CD19⁺ CD20^lo B cells were more resistant to VACV infection than CD19⁺ CD20^hi B cells (Fig. 1E), probably due to less binding of this B cell subpopulation to VACV. Thus, CD19⁺ CD20^lo B cells exhibit low susceptibility to VACV binding and infection (Fig. 1F). Taken together, VACV robustly bound to but moderately or weakly infected human primary B cells.

FIG 1 — VACV robustly bound to but moderately or weakly infected primary human B cells. Recombinant VACV strains of vA5L-YFP and VV-EGFP were used to analyze VACV binding and infection in isolated primary human B cells, respectively. VACV was used at an MOI of 0.5. (A) Representative FCM plots for VACV binding. (B) Pooled data of VACV binding to CD19⁺ CD20^hi and CD19⁺ CD20^lo B cells from 5 healthy blood donors. (C) VACV (vA5L-YFP, green) binding to lipid rafts (CTB staining, red) on the surface of *ex vivo* primary human B cells. Scale bars represent 5 μM. The data represent the results of VV binding to lipid rafts on *ex vivo* primary human B cells from 3 blood donors. (D) Representative FCM plots for VACV infection. (E) Pooled data of VACV infection of CD19⁺ CD20^hi and CD19⁺ CD20^lo B cells from 3 healthy blood donors. (F) Analysis and comparison of VACV binding and infection in CD19⁺ CD20^hi and CD19⁺ CD20^lo B cells. Graphs represent means ± standard errors of the means (SEM). Data were compared using paired t test (B and E) or Student's t test (F). *, *P <* 0.05; **, *P <* 0.01; ***, *P <* 0.001; ns, not significant. Mock, binding, or infection experiments without addition of virions are also shown.

VACV aggressively bound to multiple subsets of B cells but not plasmablasts.

We next comprehensively analyzed the susceptibility of peripheral B cells at different differentiation and maturation stages to VACV binding. Circulating peripheral blood B cells are highly heterogeneous and can be classified according to their differentiation and maturation stages into (i) transitional (CD19⁺ CD38⁺ CD24⁺ CD27⁻), (ii) mature naive (CD19⁺ IgD⁺ CD27⁻ CD21⁺), (iii) memory (CD19⁺ CD27⁺), (iv) plasmablast (CD19⁺ CD20^lo CD138⁻ CD38⁺ CD27⁺), and (v) plasma (CD19⁺ CD20⁺ CD138⁺) cells (41, 42). Among these B cell subpopulations, memory B cells can be further classified into nonswitched memory (CD19⁺ CD27⁺ IgD⁺ IgM⁺), IgM-only memory (CD19⁺ CD27⁺ IgD⁻ IgM⁺), and class-switched memory (CD19⁺ CD27⁺ IgD⁻ IgM⁻) based on their differential expression of unique Ig heavy chain of IgD or IgM isotypes (41, 42). The VACV binding and infection spectrum of these B cell subpopulations has not been studied. To study the binding spectrum, we incubated vA5L-YFP, a recombinant VACV containing viral core protein fused with yellow fluorescent protein (YFP), to allow direct visualization of virus particles by flow-cytometric analysis (FCM) or confocal microscopy with ex vivo peripheral B cells at 4°C for 30 min, a condition that allows VACV binding but not entry. After cell-free virions were removed by extensive washing, cells were subjected to FCM of VACV binding to specific B cell subsets. We found that VACV displayed robust binding to all other subsets of ex vivo B cells except plasmablasts (Fig. 2A and B). The majority of transitional, mature naive, memory, nonswitched memory, IgM memory, class-switched memory, and plasma cells were VACV positive, ranging from 56.1% of mature naive (CD19⁺ IgD⁺ CD27⁻ CD21⁺) to 81.6% of nonswitched memory (CD19⁺ CD27⁺ IgD⁺ IgM⁺) B cells (Fig. 2A and B). In contrast, less than 14.9% of plasmablasts exhibited VACV binding (Fig. 2A and B). Thus, VACV binds to plasmablasts at a significantly lower level than that of other B cell subsets.

The receptors for poxviruses have yet to be discovered. One study has suggested that myxoma virus, a rabbit-specific poxvirus that causes a lethal disease (myxomatosis) in rabbits only (32, 45), uses chemokine receptors such as CXCR4 and CCR5 to bind to and enter into cells (32, 46). However, these results have not been reproduced with other poxviruses, such as VACV. To test whether chemokine receptors serve as VACV receptors, we examined VACV binding to B cells expressing CXCR4 or CXCR5 versus CD80 or CD86. In CXCR4⁺ or CXCR5⁺ B cells, 51.9% and 58.5%, respectively, displayed VACV bound to the cell surface, while approximately 70% CD80⁺ or CD86⁺ B cells were VACV bound (Fig. 2C and D). However, these results did not indicate that CXCR4⁺ or CXCR5⁺ B cells were less sensitive to VACV binding than CD80⁺ or CD86⁺ B cells, because CXCR4 and CXCR5 were highly expressed on the surface of ex vivo B cells across different B subpopulations, whereas CD80 and CD86 were only expressed in a small percentage (6% to 10%) of ex vivo B cells (data not shown).

We next focused on the VACV-positive B cells to analyze the distribution of the specific B cell subsets bound with VACV particles. As shown in Fig. 2E, VACV-bound cells mainly consisted of memory (34.2%) and mature naive (32.6%) cells, followed by nonswitched memory, class-switched memory, transitional, and plasma cells. VACV-bound B cells contained a very small percentage (<3%) of IgM memory cells and minimal plasmablasts (<1%) (Fig. 2E). When we analyzed the phenotype of VACV-positive B cells in the context of CXCR4, CXCR5, CD80, or CD86 expression, we found that the majority of the VACV-bound B cells expressed CXCR5 (93.8%) or CXCR4 (59.8%), whereas 7.9% and 11.6% of VACV-bound B cells expressed CD80 and CD86, respectively (Fig. 2F and G). Therefore, VACV aggressively binds to the majority of B cell subpopulations, but not plasmablasts, and VACV-bound B cells express high levels of chemokine receptors, particularly CXCR5.

VACV effectively infected subpopulations of memory B cells.

Since VACV displayed a minimal binding to plasmablasts but a strong binding to other subsets of B cells, we sought to assess the VACV infection spectrum in B cell subsets. We found that memory B cells and memory B cell subsets, including nonswitched memory, IgM memory, and class-switched memory cells, were highly permissive to VACV infection, as EGFP-positive cells were observed, ranging from 58.1% of class-switched memory (CD19⁺ CD27⁺ IgD⁻ IgM⁻) to 66.4% of nonswitched memory (CD19⁺ CD27⁺ IgD⁺ IgM⁺) B cells (Fig. 3A and B). In contrast, only a small percentage of mature naïve (CD19⁺ IgD⁺ CD27⁻ CD21⁺), transitional (CD19⁺ CD38⁺ CD24⁺ CD27⁻), plasma (CD19⁺ CD20⁺ CD138⁺), and plasmablast (CD19⁺ CD20^lo CD138⁻ CD38⁺ CD27⁺) cell subsets were EGFP positive, ranging from 2.3% of transitional to 5.4% of mature naive B cells (Fig. 3A and B). Notably, memory B cells comprised less than 32% of the total B cells in our study (data not shown). Taking this into account, the robust infection (>58%) of memory B cells was diluted in other B cell subsets that were resistant to VACV infection, resulting in a small percentage of infected cells in total B cells. Interestingly, VACV binding to plasma, transitional, and mature naive B cells was robust, but few of these VACV-bound cells displayed infection (Fig. 3A and B), suggesting that these cells were sensitive to VACV binding but resistant to VACV infection. There was no difference in VACV infection between cells expressing CXCR4 and CXCR5 (Fig. 3C and D). However, B cells expressing CD86 were more sensitive to VACV infection than CD80-expressing compartments (Fig. 3C and D), suggesting that activated CD86⁺ B cells are more vulnerable to VACV infection.

We next focused on the VACV-infected B cells to analyze the distribution of the specific B cell subsets with VACV infection. As shown in Fig. 3E, VACV-infected cells mainly consisted of memory cells (66.1%), particularly class-switched memory cells (45.8%), while other B cell subsets comprised a small portion of the VACV-infected B cell population, ranging from 0.3% of plasmablasts to 12.7% of mature naive cells (Fig. 3E). This feature of cell contributions to the pool of VACV-infected primary human B cells was unlikely to be due to effects of VACV infection on B cell survival or epigenetic regulation, as the frequency of B cell subsets between VACV-infected and mock-infected conditions did not show any discernible differences (Fig. 3F). Interestingly, nearly all of the VACV-infected B cells expressed CXCR4 or CXCR5 (Fig. 3G and H), whereas only a small portion of VACV-infected cells expressed CD80 or CD86 (Fig. 3G and H). These results suggest that VACV preferentially infects memory B cells that express chemokine receptors such as CXCR4 and CXCR5.

VACV underwent an abortive infection in unstimulated primary human B cells at the late stage of VACV gene expression.

Poxviruses, such as VACV, undergo an abortive infection in primary human APCs, including monocytes and DCs, and in several human cell lines as well (47 –52). It is unclear whether VACV undergoes a productive or abortive infection in primary human B cells. To this end, we employed the virus plaque assay to evaluate VACV production in stimulated and unstimulated primary human B cells. We found that VACV titers in the cell lysates of unstimulated B cells initially increased and reached a plateau by 24 h postinfection (h.p.i.) (Fig. 4A and B). After 24 h of infection in unstimulated B cells, VACV titers started to decline (Fig. 4A and B). Similar results for viral plaque assay were also observed in the supernatant of unstimulated B cells over a period of 48 h.p.i. (data not shown). These results indicate that VACV replication is not sustainable during the infection of unstimulated B cells and leads to the overall result of an abortive infection.

FIG 4 — VACV infection in isolated *ex vivo* primary human B cells was aborted at late gene expression stage. Examination of VACV infection in unstimulated primary human B cells. (A) Representative virus plaque assay using unstimulated primary human B cells infected with VACV-WR at an MOI of 2 for 3 h, 24 h, or 48 h. (B) Pooled data of virus plaque assays from 5 healthy blood donors. Each dot in a group represents data from a single individual. (C) RT-PCR analysis of VACV early, intermediate, and late gene expression in unstimulated primary human B cells. Zero hour indicates that cells were incubated with VACV under the binding condition, followed by washing with cold PBS and isolation of total RNA. Data were obtained from 5 healthy blood donors.

We next determined which step in the life cycle of VACV infection was halted in unstimulated B cells. Since VACV binding was not the limiting step, we focused on analysis of the expression of VACV early (A23R and C11R), intermediate (A2L and G8R), and late (A17L and B7R) genes. Reverse transcription-PCR (RT-PCR) results revealed that the expression of VACV early (A23R and C11R) and intermediate (A2L and G8R) genes was detected within 1 h and 9 h of postinfection, respectively (Fig. 4C). However, the expression of VACV late genes (A17L and B7R) was minimal or not detected in infected B cells by 24 h postinfection (Fig. 4C). Thus, the abortive VACV infection in primary human B cells is most likely due to the failure to adequately express the late viral genes that are important for viral assembly, maturation, and release.

VACV underwent a productive infection in activated primary human B cells.

While naive T cells show minimal to no VACV binding and infection, activated T cells have been shown to allow for strong binding and limited viral infection (39, 44). Additionally, autoimmune skin disorders are a contraindication for receiving live VACV as the smallpox vaccine, and these disorders result in overactivation of B cells (53, 54). Thus, we examined VACV infection in activated B cells to assess the interplay between B cell activation and VACV infection. We stimulated primary human B cells with TLR9 agonist CpG ODN 2006 plus IgG/IgM antibodies to activate B cells. As shown in Fig. 5A and B, cells were activated, as the lymphocyte activation markers (CD69 and CD83) and APC costimulatory molecules (CD80 and CD86) were markedly induced on the surface of stimulated B cells. We used a multiplex assay to measure the levels of 45 cytokines, chemokines, and growth factors in the cell-free supernatants of unstimulated and stimulated B cells. We found that 33 of these cytokines, chemokines, and growth factors, including alpha interferon (IFN-α), IFN-γ, interleukin-1α (IL-1α), IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-18, IL-21, IL-27, LIF, IFN-α, IL-8, macrophage inflammatory protein 1α (MIP-1α)/CCL3, MIP-1β/CCL4, RANTES/CCL5, stromal cell-derived factor 1α (SDF-1α)/CXCL12, granulocyte macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), nerve growth factor β (NGF-β), platelet-derived growth factor (PDGF)-BB, placenta growth factor 1 (PIGF-1), stem cell factor (SCF), vascular endothelial growth factor A (VEGF-A), and VEGF-D were markedly induced at 3 h of stimulation and continued to increase along the period of 24 h of stimulation (Table 1). Enzyme-linked immunosorbent assay (ELISA) results also validated that the levels of IL-6 and IL-10 were increased in the cell-free supernatants of simulated B cells compared to that of unstimulated B cells (Fig. 5B), although increased IL-10 did not reach statistical significance due to the immense variation among samples (Fig. 5C). These results demonstrate that primary human B cells are activated in vitro.

FIG 5 — VACV infection in stimulated primary human B cells was permissive. Evaluation of VACV infection in stimulated primary human B cells. (A) Analysis of B cell activation using FCM to assess B cell activation markers, including CD69, CD80, CD83, and CD86. (B and C) ELISA measurement of IL-6 (n = 6) and IL-10 (n = 5) levels in the supernatants from unstimulated and stimulated primary human B cells. (D) Representative virus plaque assay using cell lysates of stimulated B cells infected with VACV-WR at an MOI of 2 for various time points as indicated. (E) Pooled data for virus plaque assays. Each dot in a group represents data from a single individual (n = 6). (F) Representative virus plaque assay using cell-free supernatants from stimulated B cells infected with VACV-WR at an MOI of 2 for various time points as indicated. (G) Pooled data of the virus plaque assays from cell-free supernatants of stimulated B cells infected with VACV-WR at an MOI of 2 for various time points as indicated. Each dot in a group represents data from a single individual (n = 3). (H) RT-PCR analysis of VACV early, intermediate, and late gene expression in stimulated B cells infected with VACV-WR at an MOI of 2 for various time points as indicated. The data represent the results of VACV gene expression in stimulated B cells from 3 blood donors (n = 3). Data were compared using paired t test (B and C) or ANOVA with Tukey’s *post hoc* test (E and G). Lines represent means ± SEM. *, *P <* 0.05; ***, *P <* 0.001. unstim, unstimulated B cells; stim, stimulated B cells; ns, not significant; h.p.i., hours postinfection.

TABLE 1.

Production of cytokines/chemokines/growth factors by unstimulated and stimulated B cells

Analyte	Production at^a :
	3 h		8 h		24 h
	Unstimulated	Stimulated	Unstimulated	Stimulated	Unstimulated	Stimulated
IFN-α	2.3 ± 3.3	19.9 ± 13.7*	1.9 ± 1.7	35.8 ± 21.1*	2.2 ± 2.6	47.0 ± 54.8*
IFN-γ	2.4 ± 1.7	44.0 ± 40.0*	2.2 ± 1.5	83.9 ± 71.4*	1.8 ± 1.3	79.0 ± 113.4*
IL-1α	1.0 ± 1.1	8.4 ± 5.5*	0.5 ± 0.7	11.8 ± 8.4*	0.9 ± 0.8	14.2 ± 15.8*
IL-1β	1.5 ± 1.7	38.3 ± 27.2*	1.1 ± 1.0	76.4 ± 65.4*	1.0 ± 1.8	125.2 ± 169.2*
IL-1RA	108.1 ± 122.8	1,761 ± 1,369*	165.1 ± 183.5	3,661 ± 2,915*	235.8 ± 331.4	36,391 ± 82,493*
IL-2	13.8 ± 8.7	132.8 ± 86.8*	15.6 ± 11.7	230.6 ± 192.5*	10.7 ± 5.2	200.8 ± 212.3*
IL-4	10.5 ± 6.5	75.1 ± 45.7*	9.7 ± 6.5	142.9 ± 99.1*	9.8 ± 4.3	147.0 ± 140.5*
IL-5	5.6 ± 1.8	33.1 ± 19.1*	4.7 ± 2.6	47.7 ± 27.7*	4.6 ± 3.3	33.9 ± 22.6*
IL-6	19.5 ± 30.9	748 ± 399*	27.4 ± 31.6	1,738 ± 1,261*	16.0 ± 25.6	2,921 ± 3,939*
IL-7	1.7 ± 1.4	12.1 ± 5.9*	2.0 ± 1.8	15.7 ± 7.7*	1.9 ± 1.4	18.6 ± 8.5*
IL-9	18.4 ± 35.7	18.1 ± 27.4	15.6 ± 25.0	36.8 ± 42.3	8.1 ± 18.6	83.3 ± 121.6
IL-10	1.0 ± 0.4	32.0 ± 26.2*	1.0 ± 0.3	127.8 ± 144.5*	1.0 ± 0.2	215.0 ± 146.0*
IL-12p70	0.04 ± 0.0	26.7 ± 18.5*	0.04 ± 0.0	38.2 ± 21.5*	0.04 ± 0.0	42.3 ± 36.0*
IL-13	5.2 ± 5.6	35.5 ± 19.1*	4.3 ± 5.0	53.7 ± 33.1*	2.8 ± 3.3	61.8 ± 52.3*
IL-15	8.6 ± 5.6	87.4 ± 37.3*	6.9 ± 6.4	136.0 ± 68.9*	12.1 ± 5.5	146.6 ± 124.1*
IL-17A	3.4 ± 3.1	50.6 ± 29.8*	3.9 ± 2.5	67.4 ± 44.6*	3.1 ± 1.8	67.1 ± 61.9*
IL-18	10.5 ± 6.2	54.3 ± 25.5*	12.2 ± 10.8	105.5 ± 69.2*	11.4 ± 6.5	138.7 ± 183.2*
IL-21	11.5 ± 9.4	458.9 ± 338.7*	10.7 ± 10.1	566.2 ± 356.9*	17.3 ± 11.2	411.4 ± 340.5*
IL-22	86.4 ± 96.2	95.9 ± 29.4	53.9 ± 37.0	130.6 ± 63.7	42.0 ± 33.5	138.6 ± 51.2*
IL-23	289.9 ± 351.4	334.2 ± 266.4	138.3 ± 129.6	557.2 ± 360.0	439.7 ± 418.6	701.7 ± 529.6
IL-27	59.4 ± 85.7	545.3 ± 294.9*	48.6 ± 67.6	736.1 ± 497.4*	28.0 ± 56.1	909.1 ± 909.4*
IL-31	54.6 ± 119.1	57.4 ± 64.3	39.5 ± 63.5	105.8 ± 142.5	3.3 ± 0.0	125.4 ± 138.8
LIF	0.6 ± 0.4	7.1 ± 3.5*	0.7 ± 0.4	9.2 ± 4.4*	0.7 ± 0.6	8.5 ± 5.6*
TNF-α	7.9 ± 2.9	36.1 ± 22.0*	8.0 ± 4.3	73.0 ± 57.6*	7.0 ± 2.7	82.0 ± 83.3*
TNF-β	1.6 ± 0.0	1.7 ± 0.2	1.7 ± 0.2	10.4 ± 17.7	1.6 ± 0.0	130.3 ± 226.7
Eotaxin	1.5 ± 0.2	2.9 ± 1.3	1.6 ± 0.3	5.3 ± 2.7*	1.7 ± 0.6	6.4 ± 5.0*
GROα	1.4 ± 1.6	8.3 ± 11.0	0.8 ± 0.6	28.7 ± 39.0	1.3 ± 1.3	67.1 ± 129.2*
IL-8	193.5 ± 344.9	523.8 ± 580.7*	356.8 ± 745.8	1,339 ± 1,058*	392.1 ± 786.7	2,762 ± 3,667*
IP-10	6.6 ± 4.1	8.4 ± 3.2	7.2 ± 4.3	12.0 ± 5.3	5.9 ± 6.5	18.0 ± 9.1
MCP-1	338.4 ± 424.5	719.3 ± 799.6*	307.8 ± 356.2	1,409 ± 1,415*	1,317 ± 1,469	2,191 ± 2,807
MIP-1α	3.6 ± 3.0	65.7 ± 35.5*	5.2 ± 6.0	123.1 ± 72.3*	5.8 ± 2.9	274.3 ± 414.8*
MIP-1β	23.7 ± 32.4	174.4 ± 54.3*	18.8 ± 23.2	393.1 ± 125.4*	52.7 ± 17.6	841.3 ± 620.6*
RANTES	6.2 ± 8.9	4.7 ± 3.5	4.9 ± 4.8	16.8 ± 26.0*	3.7 ± 3.6	19.7 ± 19.1*
SDF-1α	394.0 ± 528.0	1,777 ± 1,581*	197.1 ± 212.8	3,377 ± 2,579*	806.0 ± 915.1	6,279 ± 9,814*
BDNF	2.3 ± 2.5	3.5 ± 1.9	2.0 ± 2.0	5.3 ± 2.8	2.1 ± 2.3	8.2 ± 10.8*
EGF	3.6 ± 4.0	8.1 ± 2.6	2.6 ± 1.5	10.7 ± 4.1*	3.9 ± 4.3	13.2 ± 9.1
FGF-2	6.1 ± 8.2	8.7 ± 8.7	7.1 ± 7.2	12.5 ± 14.3	5.3 ± 4.3	14.0 ± 16.4
GM-CSF	13.1 ± 13.4	168.8 ± 78.1*	20.2 ± 22.9	197.3 ± 111.0*	9.3 ± 10.2	180.3 ± 126.2*
HGF	8.0 ± 5.7	86.1 ± 46.7*	7.0 ± 4.4	127.0 ± 79.8*	9.1 ± 4.4	162.3 ± 139.8*
NGFβ	2.5 ± 1.5	20.9 ± 12.7*	4.2 ± 2.1	42.8 ± 30.7*	2.9 ± 2.1	64.6 ± 88.3*
PDGF-BB	8.4 ± 6.0	13.9 ± 5.4	5.8 ± 1.8	15.9 ± 6.4*	7.1 ± 3.7	13.8 ± 5.7*
PIGF-1	3.4 ± 4.4	58.0 ± 35.3*	2.2 ± 3.1	93.1 ± 60.1*	3.5 ± 3.5	81.2 ± 70.8*
SCF	3.7 ± 3.1	32.6 ± 22.6*	2.5 ± 2.9	56.0 ± 41.4*	2.5 ± 2.1	62.9 ± 69.4*
VEGF-A	11.9 ± 15.9	470.5 ± 324.9*	8.0 ± 10.8	607.0 ± 328.1*	10.0 ± 11.5	454.0 ± 266.5*
VEGF-D	2.3 ± 2.4	42.3 ± 20.3*	1.6 ± 0.8	47.7 ± 32.1*	3.4 ± 3.9	41.2 ± 31.8*

Open in a new tab

Data are represented as means ± SD (standard deviations), n= 5. *, P < 0.05 for comparison between unstimulated and stimulated primary human cells. Mann-Whitney test was used to compare the production of cytokines, chemokines, and growth factors by stimulated versus unstimulated B cells at each time point.

To assess the replication and productiveness of VACV infection in stimulated B cells, we used the viral plaque assay to quantitate VACV virions in infected cells and cell-free supernatant of activated B cells infected with VACV. In the cell lysates of VACV-infected stimulated B cells, viral titers displayed an increase over a period of 48 h.p.i. (Fig. 5D and E). In the supernatants, however, viral titers reduced over time of cell culture (Fig. 5F and G). These results suggested that new virions were generated but were delayed to egress into culture supernatants. Additionally, assessment of VACV gene expression in infected stimulated B cells showed expression of early, intermediate, and late viral genes (Fig. 5H), further indicating that activated B cells support productive VACV infection.

To study whether B cell function was affected by VACV infection, we used the multiplex assay to analyze and compare the production of 45 cytokines, chemokines, and growth factors in the cell-free supernatants from VACV-infected versus mock-infected unstimulated and activated B cells. In general, the production of these cytokines was not significantly affected by VACV infection (data not shown). However, several cytokines, such as IL-10 and IL-12p70 (data not shown), displayed a trend of decreased production (data not shown). Thus, VACV binding and infection do not profoundly affect the activities of ex vivo or activated B cells in the context of their biosynthesis of cytokines, chemokines, and growth factors.

DISCUSSION

In the present study, we systematically studied VACV binding to and infection of unstimulated and stimulated primary human B cells at different differentiation and maturation stages. We found that VACV robustly bound to but moderately or weakly infected ex vivo primary human B cells (Fig. 1), which is in agreement with previous reports using PBMCs as targets cells of VACV binding and infection (36, 38, 39, 44). These results suggest that B cells express VACV receptors on their surface but have cellular factors or signaling pathways that restrict VACV uptake, entry, and replication. Additionally, our study revealed that the plasmablast compartment was the only subset of human primary B cells that displayed resistance to VACV binding, while the other subsets of B cells, including transitional, mature naive, memory, and plasma cells, displayed high VACV binding activities that are similar to those of other APCs (Fig. 2). These results indicate that plasmablasts lack expression of viral receptors or attachment factors necessary for the initial encounter between host cell and VACV. While many viruses, such as influenza A virus and hepatitis A virus (HAV), use a single molecular species as their receptors (55), several viruses, such as hepatitis B virus (HBV), gain their access into target cells through subsequent engagements of specific proteins, glycoproteins, or carbohydrate residues on the surface of the host cells. Given that VACV effectively uses cell surface glycosaminoglycan (GAG)- and heparin sulfate-dependent and -independent pathways to initiate viral infection (47, 56 –60), VACV most likely uses a multiprotein receptor-entry-fusion complex to initiate viral binding, attachment, entry, and infection. We found that primary human B cells at different maturation and activation stages exhibited differential activities for VACV binding and infection and thereby could represent natural cellular models to identify and study VACV receptors that have not yet been identified. The various B cell subsets could also be a useful tool to dissect the molecular mechanisms involved in VACV binding, penetration, entry, and replication. This knowledge is essential for the development of a safer smallpox vaccine and more efficacious VACV-based vaccines against cancer and infectious pathogens. VACV is the most widely used vector for vaccine development (30). Currently, many clinical trials are evaluating the use of VACV vectors as preventative and therapeutic vaccines to target several types of cancer and various pathogens, such as HIV-1, HBV, influenza, malaria, and tuberculosis (22 –30). In addition, poxviruses are being implemented as vaccine vectors for various veterinary vaccines (19). The poxvirus-based vaccine success for cancer and infectious diseases in both humans and animals has led to a revival of interest in characterizing poxvirus binding and infection tropism.

Several cellular proteins, such as epidermal growth factor receptor (EGFR) and chemokine receptors, play roles as viral receptors to mediate poxvirus binding and entry (46, 61). In this study, we studied the role of chemokine receptors CXCR4 and CXCR5 in VACV binding. We observed that the vast majority (93.8%) of VACV-bound cells were positive for CXCR5 (Fig. 2F and G), while a substantial portion (59.8%) of VACV-bound cells was positive for CXCR4 (Fig. 2F and G), indicating that both CXCR5 and CXCR4 are associated with VACV binding. CXCR4 has been demonstrated to be one of the chemokine receptors for poxvirus (myxoma virus) binding and entry (46). Our study demonstrates that CXCR5 likely also plays an important role in VACV binding, thereby representing a new VACV receptor candidate. Since CXCR5 and CXCR4 are widely expressed on a variety of cell types in humans and animals, their coexpression on the cell surface may also be an important determinant for VACV binding. Notably, despite the high percentage of VACV-bound CXCR4⁺ or CXCR5⁺ B cells, only a fraction of these CXCR4⁺ or CXCR5⁺ cells was infected (Fig. 3D). These results suggest that VACV uses chemokine receptors, particularly CXCR5, as receptors for virus binding but requires other cellular proteins or signaling pathways for virus entry and replication.

In unstimulated ex vivo B cells, VACV displayed an abortive infection. To understand the mechanisms underlying the abortive infection, we analyzed the expression of VACV early, intermediate, and late genes in the infected B cells. We found that the expression of early (C11R and A23R) and intermediate (A2L and G8R) viral genes occurred, but the expression of late genes (A17L and B7R) was not detected (Fig. 4). These results indicate that the abortive VACV infection in unstimulated ex vivo primary human B cells occurs at the late viral gene expression stage. These findings provide insights into and explanations of previous study results that revealed an abortive VACV infection in B cells of PBMCs (39). VACV is a large enveloped virus containing a linear double-stranded DNA genome of ∼192 kb in length that encodes approximately 200 viral proteins. These open reading frames (ORFs) are closely spaced and temporally regulated (62, 63). To date, there are 38 VACV genes that have been identified as late genes (62, 63). These late genes are transcribed following genome replication and require the successive synthesis of intermediate and late transcription factors, encoded by early and intermediate genes, respectively (62, 63). In addition, cellular factors are also directly involved in regulation of late gene expression (62, 63). Therefore, a genome-wide sequencing of VACV-sensitive and -resistant B cells to study early, intermediate, and late transcription will provide a better understanding of poxvirus replication in primary human cells and lead to improvements in design and development of expression vectors and recombinant vaccines.

Live VACV vaccine induces strong humoral responses that play a crucial role in protection against smallpox (64 –66). After vaccination with live VACV, strong antibody responses against VACV surface proteins, core proteins, and soluble proteins are elicited (64), demonstrating that the human anti‐VACV antibody response is broad. Importantly, there is a correlation between pre‐existing serum neutralizing antibody titers and protection against contracting smallpox (67, 68). These findings indicate that VACV infection generated long-lived virus-specific memory B cells or directly interacts with prememory or memory B cells. We demonstrated that primary human memory B cells, including nonswitched, class-switched, and IgM memory B cells, were highly susceptible to VACV binding and infection (Fig. 2 and 3), suggesting that VACV is able to directly interact with memory B cells. However, the contributions of VACV and memory B cell interactions to protective immunity against smallpox remain unknown. Better understanding of the interplay between VACV and memory B cells may be critical for dissecting the mechanisms underlying the high efficacy, immunogenicity, and safety of live VACV-based vaccines against smallpox and other pathogens, such as HIV. To date, hundreds of different HIV vaccine candidates have been generated, and over 40 of them have been tested in thousands of volunteers worldwide. Poxvirus-based HIV vaccine has been the only one that has demonstrated preventive efficacy from a large-scale HIV vaccine trial (known as RV144) (10, 11). The prespecified analysis of immune correlates of risk has shown that anti-HIV gp120 antibodies, particularly the IgG1 and IgG3 subclasses, seem to play a predominant role in protection against HIV acquisition (69). It would be very interesting to study whether the R144 immune protection against HIV acquisition is related to the direct effects of VACV on memory B cells in vivo.

While naive resting T cells show minimal to no VACV binding and infection, activated T cells have been shown to allow for strong binding and limited viral infection (39, 44). Thus, we examined VACV infection in activated B cells to assess the interplay between B cell activation and VACV infection. We stimulated primary human B cells with TLR9 agonist CpG ODN 2006 plus IgG/IgM antibodies to activate B cells. Cell-free supernatants from unstimulated and stimulated B cells were subjected to a multiplex assay to determine the concentration of 45 cytokines, chemokines, and growth factors. We found that the vast majority of these cytokines, chemokines, and growth factors were markedly induced at 3 h of stimulation and continued to increase along the period of 24 h of stimulation (Table 1). However, neither VACV binding nor infection profoundly affected the production of these cytokines, chemokines, and growth factors by unstimulated and stimulated B cells. This result was in line with the effects of VACV infection on the frequency of B cell subsets. As shown in Fig. 3F, comparison of the frequency of B cell subsets between VACV-infected versus mock-infected cells did not show any discernible differences. However, we found that upon stimulation, B cells displayed increased VACV infection compared to that of unstimulated compartments (Fig. 4), and VACV infection was no longer abortive (Fig. 5E to H). This result indicates that activation of B cells leads to the synthesis of essential receptors or signaling molecules necessary for VACV entry and replication. Since the poxvirus infection is less restricted in activated B cells, this could be one factor leading to the uncontrolled response to VACV infection in individuals with autoimmune skin disorders, which lead to the production of overactive B cells. In combination with the ability to use unstimulated B cells as a tool to study VACV binding and entry, the ability to use activated B cells to better understand the mechanisms underlying VACV infection permissiveness demonstrate that primary B cells may prove to be a vital tool to help understand VACV infection in the hopes of developing better oncolytic poxviruses.

MATERIALS AND METHODS

Ethics statement.

Peripheral blood samples from healthy blood donors were obtained from the Indiana Blood Center (Indianapolis, IN) under an IRB protocol approved by the Indiana University School of Medicine Institutional Review Board for Human Research (Indianapolis, IN). Written informed consent was obtained from each participant before specimen collection.

Preparation and activation of primary human B cells.

Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood or leukapheresis products using gradient centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech AB, Uppsala, Sweden) (47). Isolated PBMCs were subjected to immunomagnetic positive selection of B cells using human CD19 microbeads (Miltenyi Biotec, Auburn, CA). Resulting cell preparations contained more than 97% purity of the B cells when assessed by CD19 staining of an alternate epitope and flow-cytometric analysis (FCM).

Purified ex vivo B cells were directly used for VACV binding and infection. Purified B cells were also activated by culturing in the presence of 20 μg/ml AffiniPure F(ab′)₂ fragment goat antihuman IgG + IgM (H+L) (Jackson Immunoresearch Laboratories, West Grove, PA) and 50 nM TLR9 agonist CpG ODN 2006 (InvivoGen, San Diego, CA) in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% streptomycin-penicillin, and 2 mM l-glutamine (complete RPMI 1640 medium) for 3 to 24 h at 37°C. Supernatants were subjected to multiplex immunoassays and enzyme-linked immunosorbent assays (ELISAs) to measure the production of cytokines, chemokines, and growth factors, while cells were subjected to surface staining with antibodies against human CD69, CD80, CD83, and CD86 to evaluate B cell activation. Activated B cells were tested for their susceptibility to VACV binding and infection.

Antibodies and FCM.

Anti-human monoclonal antibodies conjugated with fluorochromes were purchased from BD Biosciences (San Jose, CA) or BioLegend (San Diego, CA). These fluorochrome-conjugated antibodies included BCMA^PE, CCR6^BV605, CCR7^PE, CD10^PerCP-Cy5.5, CD20^APC-Cy7, CD21^AF647, CD23^PE, CD24^BV605, CD25^BV605, CD27^BV510, CD38^BV421, CD40^PE-Cy7, CD45RA^BV605, CD80^PE-Cy7, CD86^PE, CD138^PE-Cy7, CD226^AF647, CXCR4^PE-Cy7, CXCR5^AF647, HLA-DR^PerCP-Cy5.5, IgD^AF700, IgM^PerCP-Cy5.5, and TACI^AF647. These antibodies and matched-isotype control antibodies were used for cell surface staining of isolated B cells to analyze B cell phenotype (Table 2). The fluorochrome-conjugated antibodies, including CD69^FITC, CD83^PE, CD80^PE-Cy7, and CD86^BV510, were used for cell surface staining of unstimulated and stimulated B cells to evaluate B cell activation. Appropriate isotype controls were used at the same protein concentration as the test antibodies for control staining, which was performed during every FCM. Propidium iodide (PI) staining was also performed in each FCM to exclude dead cells that were PI positive. After cell surface staining, B cells were subjected to FCM using a BD LSRFortessa (BD Bioscience, San Diego, CA). The data were analyzed using FlowJo software (Tree Star, San Carlos, CA).

TABLE 2.

Antibodies and staining strategy for flow-cytometric analysis of B cell phenotypes

Panel 1	Panel 2	Panel 3	Panel 4
CCR7^PE	CD23^PE	CD86^PE	BCMA^PE
PI	PI	PI	PI
HLA-DR^PerCP-Cy5.5	CD69^PerCP-Cy5.5	IgM^PerCP-Cy5.5	CD10^PerCP-Cy5.5
CXCR4^PE-Cy7	CD40^PE-Cy7	CD80^PE-Cy7	CD138^PE-Cy7
CXCR5^AF647	CD21^AF647	CD226^AF647	TACI^AF647
IgD^AF700	IgD^AF700	IgD^AF700	IgD^AF700
CD20^APC-Cy7	CD20^APC-Cy7	CD20^APC-Cy7	CD20^APC-Cy7
CD38^BV421	CD38^BV421	CD38^BV421	CD38^BV421
CD27^BV510	CD27^BV510	CD27^BV510	CD27^BV510
CCR6^BV605	CD25^BV605	CD45RA^BV605	CD24^BV605

Open in a new tab

VACV preparation, binding, and infection.

VV-EGFP and vA5L-YFP, two recombinant reporter viruses generated on the background of Western Reserve VACV (VACV-WR) strain, were obtained from J. W. Yewdell and B. Moss (NIH, Bethesda, MD). VV-EGFP contains a chimeric gene encoding the influenza virus nucleoprotein, the ovalbumin SIINFEKL peptide, and EGFP regulated by the P7.5 early/late promoter (70). EGFP expression was used in this study to evaluate VACV infection. The vA5L-YFP virus contains the VACV A5L core protein fused with yellow fluorescence protein (YFP), allowing for direct visualization of VACV particles by fluorescence microscopy or FCM (71). Therefore, direct visualization of vA5L-YFP was used in this study to evaluate VACV binding.

Viral stocks of VV-EGFP, vA5L-YFP, and VACV-WR were prepared as previously described (72). Briefly, VV-EGFP, vA5L-YFP, and VACV-WR were grown in the monkey kidney fibroblast cell line CV-1 (ATCC, Manassas, VA) in complete RPMI 1640 medium. Cell-associated mature virions were purified using a 36% sucrose gradient followed by a 24% to 40% sucrose gradient. The viral stocks were titrated using a viral plaque assay in CV-1 cells as previously described (72). Briefly, CV-1 cells were grown in 12-well plates to 90% confluence and overlaid with various dilutions of purified virions. After 1.5 h of incubation, cells were washed and overlaid with complete RPMI 1640 containing 1% carboxylmethyl cellulose (CMC) to prevent virus spread. Cells were cultured for 3 days and then stained with 0.01% crystal violet in 15% ethanol solution for 30 min, followed by washing so that viral plaques could be counted to calculate virus PFU.

To assess VACV binding to ex vivo B cells, isolated B cells were incubated with vA5L-YFP virions at a multiplicity of infection (MOI) of 0.5 at 4°C for 30 min in complete RPMI 1640 medium, a condition that permits only virus binding but not entry (39, 72, 73). After multiple washes with ice-cold phosphate-buffered saline (PBS), cells were fixed with 1% paraformaldehyde (PFA) and subsequently subjected to FCM. VV-EGFP was used to observe VACV infection by incubating isolated B cells with virions at an MOI of 0.5 under the binding conditions described above. Cells bound with virions were subsequently incubated at 37°C in 5% CO₂ for various periods of time. Cells were collected, fixed with 1% PFA, and subjected to FCM. Infection intensity was calculated by the percentage of EGFP-positive cells.

Confocal microscopy analysis of VACV binding.

Confocal microscopy analysis of VACV binding to ex vivo B cells was conducted as previously described (44). Briefly, ex vivo B cells were incubated with cholera toxin subunit B (CTB) conjugated with Alexa Fluor 647 (Life Technologies, Carlsbad, CA) at 4°C for 20 min to stain ganglioside M1 (GM1)-containing lipid rafts. After washing, cells were incubated with 10 PFU/cell of vA5L-YFP for 30 min under the binding conditions described above. Cells were adhered to poly-l-lysine-coated coverslips and mounted onto glass slides using ProLong Gold antifade reagent (Life Technologies, Carlsbad, CA) containing 4’,6-diamidino-2-phenylindole (DAPI) dye for fluorescent staining of DNA content and nuclei. Cells were analyzed using an Olympus FV1000-MPE confocal/multiphoton microscope fitted with a 60× objective. A z-series of images was collected using the open source software FluoRender 2.7 (University of Utah) and ImageJ 1.44p (NIH, Bethesda, MD).

RT-PCR analysis of VACV gene expression.

Stimulated or unstimulated primary human B cells were incubated with VACV-WR at an MOI of 2 at 4°C for 1 h, washed with PBS, and cultured for various periods of time in complete RPMI 1640 medium. Cells were harvested, washed with PBS, and subjected to RNA extraction using the RNeasy Minikit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA was subjected to cDNA synthesis using the Superscript III first-strand synthesis kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. RT-PCR was performed using the Taq PCR master mix kit (Qiagen, Hilden, Germany) with primers against VACV early (A23R and C11R), intermediate (A2L and G8R), and late (A17L and B7R) genes, including the following primers: A23R forward, 5′-CGTTAGTAACGCCATATGGATAATCTATTTACC-3′; A23R reverse, 5′-ACCCTAGTCGTTGGATCCATTTCTGAATC-3′; C11R forward, 5′-CAGATCATTCGCCGATAGTGGTAAC-3′; C11R reverse, 5′-GGTAGTTTAGTTCGTCGAGTGAACCT-3′; A2L forward, 5′-TCGTGTCCATAATCCTCTACCAT-3′; A2L reverse, 5′-TCCACGGATGATGTAGATGCAAA-3′; G8R forward, 5′-GGCGGATCTGTAAACATTTGGG-3′; G8R reverse, 5′-TCCTCGTAGTTTGTTGAGAGACG-3′; A17L forward, 5′-GGGCCATGGCTTATTTAAGATATTACAATATGCTT-3′; A17L reverse, 5′-GGGGGATCCTTAATAATCGTCAGTATTTAAACT-3′; B7R forward, 5′-TATCGGATCCAATAATGAGTACACTCCG-3′; B7R reverse, 5′-GAGCGAATTCTTAAAAATCATATTTTGA-3′ (Life Technologies, Carlsbad, CA). Expression data were analyzed for the presence of gene expression at particular time points of incubation as an indication of VACV infection progression.

Multiplex immunoassays and ELISAs.

Unstimulated and stimulated B cells were left uninfected or were infected with VACV for various time intervals. Cell-free supernatants were subjected to the multiplex immunoassays to simultaneously measure the concentrations of 45 human cytokines, chemokines, and growth factors (brain-derived neurotrophic factor [BDNF], Eotaxin/CCL11, EGF, fibroblast growth factor 2 [FGF-2], GM-CSF, GRO-α/CXCL1, HGF, NGF-β, leukemia inhibitory factor [LIF], IFN-α, IFN-γ, IL-1β, IL-1α, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17A, IL-18, IL-21, IL-22, IL-23, IL-27, IL-31, IP-10/CXCL10, MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, RANTES/CCL5, SDF-1α/CXCL12, TNF-α, TNF-β/LTA, PDGF-BB, PLGF-1, SCF, VEGF-A, and VEGF-D) using the ProcartaPlex human cytokine/chemokine/growth factor panel 1 45plex kit (Invitrogen, Carlsbad, CA) per the manufacturer’s instructions. The standards were run on each plate in duplicate, and all samples were assayed concurrently to avoid interassay variability. Data were acquired using a Luminex-100 system, and the concentrations of cytokines/chemokines/growth factors were calculated using the Bio-Plex Manager v6.1 software (Bio-Rad, Hercules, CA). For statistical analyses, values below the detection limit of the assay were replaced with the minimal detectable concentrations for each analyte as provided by the manufacturer.

To validate the multiplex immunoassay results, concentrations of IL-6 and IL-10 in the cell-free supernatant of cultured B cells were also measured using the human IL-6 DuoSet (R&D Systems, Minneapolis, MN) and IL-10 (Life Technologies, Carlsbad, CA) ELISA kits according to the manufacturer’s instructions.

Statistical analysis.

Data from two groups were analyzed using the Student's t test or paired t test, and data from three or more groups were analyzed using analysis of variance (ANOVA) with Tukey’s post hoc test. The Mann-Whitney test was used to compare the production of cytokines, chemokines, and growth factors by stimulated versus unstimulated B cells at each time point. P values of <0.05 were considered statistically significant.

ACKNOWLEDGMENTS

We thank J. W. Yewdell and B. Moss at NIH (Bethesda, MD) for VV-EGFP and vA5L-YFP.

This work was supported in part by a Grand Challenges Explorations (GCE) Phase II grant through the Bill & Melinda Gates Foundation (OPP1035237 to Q.Y.), NIH grant 1R21AI104268 (to Q.Y.), the Showalter Research Trust Fund (to Q.Y.), and Research Facilities Improvement Program grant C06 RR015481-01 from the National Center for Research Resources, NIH, to the Indiana University School of Medicine.

N.S. and Q.Y. designed and performed research, analyzed data, and wrote the paper. J.L., W.L., and S.R. contributed vital new reagents and assisted with virus preparation.

We declare that we have no conflicts of interest.

REFERENCES

1.Blendon RJ, DesRoches CM, Benson JM, Herrmann MJ, Taylor-Clark K, Weldon KJ. 2003. The public and the smallpox threat. N Engl J Med 348:426–432. doi: 10.1056/NEJMsa023184. [DOI] [PubMed] [Google Scholar]
2.Frey SE, Belshe RB. 2004. Poxvirus zoonoses–putting pocks into context. N Engl J Med 350:324–327. doi: 10.1056/NEJMp038208. [DOI] [PubMed] [Google Scholar]
3.Essbauer S, Pfeffer M, Meyer H. 2010. Zoonotic poxviruses. Vet Microbiol 140:229–236. doi: 10.1016/j.vetmic.2009.08.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Silva Gomes JA, de Araújo FF, de Souza Trindade G, Quinan BR, Drumond BP, Ferreira JMS, Mota BEF, Nogueira ML, Kroon EG, Santos Abrahão J, Côrrea-Oliveira R, da Fonseca FG. 2012. Immune modulation in primary vaccinia virus zoonotic human infections. Clin Dev Immunol 2012:974067. doi: 10.1155/2012/974067. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Sejvar JJ, Chowdary Y, Schomogyi M, Stevens J, Patel J, Karem K, Fischer M, Kuehnert MJ, Zaki SR, Paddock CD, Guarner J, Shieh WJ, Patton JL, Bernard N, Li Y, Olson VA, Kline RL, Loparev VN, Schmid DS, Beard B, Regnery RR, Damon IK. 2004. Human monkeypox infection: a family cluster in the midwestern United States. J Infect Dis 190:1833–1840. doi: 10.1086/425039. [DOI] [PubMed] [Google Scholar]
6.Vorou RM, Papavassiliou VG, Pierroutsakos IN. 2008. Cowpox virus infection: an emerging health threat. Curr Opin Infect Dis 21:153–156. doi: 10.1097/QCO.0b013e3282f44c74. [DOI] [PubMed] [Google Scholar]
7.Chan WM, McFadden G. 2014. Oncolytic poxviruses. Annu Rev Virol 1:119–141. doi: 10.1146/annurev-virology-031413-085442. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Nichols AC, Yoo J, Um S, Mundi N, Palma DA, Fung K, Macneil SD, Koropatnick J, Mymryk JS, Barrett JW. 2014. Vaccinia virus outperforms a panel of other poxviruses as a potent oncolytic agent for the control of head and neck squamous cell carcinoma cell lines. Intervirology 57:17–22. doi: 10.1159/000353854. [DOI] [PubMed] [Google Scholar]
9.Mejías-Pérez E, Carreño-Fuentes L, Esteban M. 2018. Development of a safe and effective vaccinia virus oncolytic vector WR-Delta4 with a set of gene deletions on several viral pathways. Mol Ther Oncolytics 8:27–40. doi: 10.1016/j.omto.2017.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Amara RR, Villinger F, Altman JD, Lydy SL, O'Neil SP, Staprans SI, Montefiori DC, Xu Y, Herndon JG, Wyatt LS, Candido MA, Kozyr NL, Earl PL, Smith JM, Ma HL, Grimm BD, Hulsey ML, Miller J, McClure HM, McNicholl JM, Moss B, Robinson HL. 2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292:69–74. doi: 10.1126/science.1058915. [DOI] [PubMed] [Google Scholar]
11.Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C, de Souza M, Adams E, Benenson M, Gurunathan S, Tartaglia J, McNeil JG, Francis DP, Stablein D, Birx DL, Chunsuttiwat S, Khamboonruang C, Thongcharoen P, Robb ML, Michael NL, Kunasol P, Kim JH, MOPH-TAVEG Investigators. 2009. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 361:2209–2220. doi: 10.1056/NEJMoa0908492. [DOI] [PubMed] [Google Scholar]
12.Weiss S, Yitzhaki S, Shapira SC. 2015. Lessons to be learned from recent biosafety incidents in the United States. Isr Med Assoc J 17:269–273. [PubMed] [Google Scholar]
13.Falendysz EA, Lopera JG, Doty JB, Nakazawa Y, Crill C, Lorenzsonn F, Kalemba LN, Ronderos MD, Mejia A, Malekani JM, Karem K, Carroll DS, Osorio JE, Rocke TE. 2017. Characterization of monkeypox virus infection in African rope squirrels (Funisciurus sp.). PLoS Negl Trop Dis 11:e0005809. doi: 10.1371/journal.pntd.0005809. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Chen N, Li G, Liszewski MK, Atkinson JP, Jahrling PB, Feng Z, Schriewer J, Buck C, Wang C, Lefkowitz EJ, Esposito JJ, Harms T, Damon IK, Roper RL, Upton C, Buller RM. 2005. Virulence differences between monkeypox virus isolates from West Africa and the Congo basin. Virology 340:46–63. doi: 10.1016/j.virol.2005.05.030. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Damaso CR, Esposito JJ, Condit RC, Moussatche N. 2000. An emergent poxvirus from humans and cattle in Rio de Janeiro State: Cantagalo virus may derive from Brazilian smallpox vaccine. Virology 277:439–449. doi: 10.1006/viro.2000.0603. [DOI] [PubMed] [Google Scholar]
16.Dhar AD, Werchniak AE, Li Y, Brennick JB, Goldsmith CS, Kline R, Damon I, Klaus SN. 2004. Tanapox infection in a college student. N Engl J Med 350:361–366. doi: 10.1056/NEJMoa031467. [DOI] [PubMed] [Google Scholar]
17.Stich A, Meyer H, Kohler B, Fleischer K. 2002. Tanapox: first report in a European traveller and identification by PCR. Trans R Soc Trop Med Hyg 96:178–179. doi: 10.1016/s0035-9203(02)90295-6. [DOI] [PubMed] [Google Scholar]
18.Kolhapure RM, Deolankar RP, Tupe CD, Raut CG, Basu A, Dama BM, Pawar SD, Joshi MV, Padbidri VS, Goverdhan MK, Banerjee K. 1997. Investigation of buffalopox outbreaks in Maharashtra State during 1992-1996. Indian J Med Res 106:441–446. [PubMed] [Google Scholar]
19.Gerdts V, Mutwiri GK, Tikoo SK, Babiuk LA. 2006. Mucosal delivery of vaccines in domestic animals. Vet Res 37:487–510. doi: 10.1051/vetres:2006012. [DOI] [PubMed] [Google Scholar]
20.Yamada T, Hamano Y, Hasegawa N, Seo E, Fukuda K, Yokoyama KK, Hyodo I, Abei M. 2018. Oncolytic virotherapy and gene therapy strategies for hepatobiliary cancers. Curr Cancer Drug Targets 18:188–201. doi: 10.2174/1568009617666170330123841. [DOI] [PubMed] [Google Scholar]
21.Park SH, Breitbach CJ, Lee J, Park JO, Lim HY, Kang WK, Moon A, Mun JH, Sommermann EM, Maruri Avidal L, Patt R, Pelusio A, Burke J, Hwang TH, Kirn D, Park YS. 2015. Phase 1b trial of biweekly intravenous Pexa-Vec (JX-594), an oncolytic and immunotherapeutic vaccinia virus in colorectal cancer. Mol Ther 23:1532–1540. doi: 10.1038/mt.2015.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Gómez CE, Nájera JL, Perdiguero B, García-Arriaza J, Sorzano COS, Jiménez V, González-Sanz R, Jiménez JL, Muñoz-Fernández MA, López Bernaldo de Quirós JC, Guardo AC, García F, Gatell JM, Plana M, Esteban M. 2011. The HIV/AIDS vaccine candidate MVA-B administered as a single immunogen in humans triggers robust, polyfunctional, and selective effector memory T cell responses to HIV-1 antigens. J Virol 85:11468–11478. doi: 10.1128/JVI.05165-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Cavenaugh JS, Awi D, Mendy M, Hill AV, Whittle H, McConkey SJ. 2011. Partially randomized, non-blinded trial of DNA and MVA therapeutic vaccines based on hepatitis B virus surface protein for chronic HBV infection. PLoS One 6:e14626. doi: 10.1371/journal.pone.0014626. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Berthoud TK, Hamill M, Lillie PJ, Hwenda L, Collins KA, Ewer KJ, Milicic A, Poyntz HC, Lambe T, Fletcher HA, Hill AV, Gilbert SC. 2011. Potent CD8+ T-cell immunogenicity in humans of a novel heterosubtypic influenza A vaccine, MVA-NP+M1. Clin Infect Dis 52:1–7. doi: 10.1093/cid/ciq015. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Porter DW, Thompson FM, Berthoud TK, Hutchings CL, Andrews L, Biswas S, Poulton I, Prieur E, Correa S, Rowland R, Lang T, Williams J, Gilbert SC, Sinden RE, Todryk S, Hill AV. 2011. A human Phase I/IIa malaria challenge trial of a polyprotein malaria vaccine. Vaccine 29:7514–7522. doi: 10.1016/j.vaccine.2011.03.083. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Tameris MD, Hatherill M, Landry BS, Scriba TJ, Snowden MA, Lockhart S, Shea JE, McClain JB, Hussey GD, Hanekom WA, Mahomed H, McShane H. 2013. Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet 381:1021–1028. doi: 10.1016/S0140-6736(13)60177-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Rahal A, Musher B. 2017. Oncolytic viral therapy for pancreatic cancer. J Surg Oncol 116:94–103. doi: 10.1002/jso.24626. [DOI] [PubMed] [Google Scholar]
28.Jebar AH, Errington-Mais F, Vile RG, Selby PJ, Melcher AA, Griffin S. 2015. Progress in clinical oncolytic virus-based therapy for hepatocellular carcinoma. J Gen Virol 96:1533–1550. doi: 10.1099/vir.0.000098. [DOI] [PubMed] [Google Scholar]
29.Downs-Canner S, Guo ZS, Ravindranathan R, Breitbach CJ, O'Malley ME, Jones HL, Moon A, McCart JA, Shuai Y, Zeh HJ, Bartlett DL. 2016. Phase 1 study of intravenous oncolytic poxvirus (vvDD) in patients with advanced solid cancers. Mol Ther 24:1492–1501. doi: 10.1038/mt.2016.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Ura T, Okuda K, Shimada M. 2014. Developments in viral vector-based vaccines. Vaccines 2:624–641. doi: 10.3390/vaccines2030624. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Moss B. 1996. Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc Natl Acad Sci U S A 93:11341–11348. doi: 10.1073/pnas.93.21.11341. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.McFadden G. 2005. Poxvirus tropism. Nat Rev Microbiol 3:201–213. doi: 10.1038/nrmicro1099. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Wein LM, Wu JT, Kirn DH. 2003. Validation and analysis of a mathematical model of a replication-competent oncolytic virus for cancer treatment: implications for virus design and delivery. Cancer Res 63:1317–1324. [PubMed] [Google Scholar]
34.Carroll MW, Moss B. 1997. Poxviruses as expression vectors. Curr Opin Biotechnol 8:573–577. doi: 10.1016/S0958-1669(97)80031-6. [DOI] [PubMed] [Google Scholar]
35.Smith GL, Moss B. 1983. Infectious poxvirus vectors have capacity for at least 25,000 base pairs of foreign DNA. Gene 25:21–28. doi: 10.1016/0378-1119(83)90163-4. [DOI] [PubMed] [Google Scholar]
36.Yu Q, Jones B, Hu N, Chang H, Ahmad S, Liu J, Parrington M, Ostrowski M. 2006. Comparative analysis of tropism between canarypox (ALVAC) and vaccinia viruses reveals a more restricted and preferential tropism of ALVAC for human cells of the monocytic lineage. Vaccine 24:6376–6391. doi: 10.1016/j.vaccine.2006.06.011. [DOI] [PubMed] [Google Scholar]
37.Hu N, Yu R, Shikuma C, Shiramizu B, Ostrwoski MA, Yu Q. 2009. Role of cell signaling in poxvirus-mediated foreign gene expression in mammalian cells. Vaccine 27:2994–3006. doi: 10.1016/j.vaccine.2009.02.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Sanchez-Puig JM, Sanchez L, Roy G, Blasco R. 2004. Susceptibility of different leukocyte cell types to vaccinia virus infection. Virol J 1:10. doi: 10.1186/1743-422X-1-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Chahroudi A, Chavan R, Kozyr N, Koyzr N, Waller EK, Silvestri G, Feinberg MB. 2005. Vaccinia virus tropism for primary hematolymphoid cells is determined by restricted expression of a unique virus receptor. J Virol 79:10397–10407. doi: 10.1128/JVI.79.16.10397-10407.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Edghill-Smith Y, Golding H, Manischewitz J, King LR, Scott D, Bray M, Nalca A, Hooper JW, Whitehouse CA, Schmitz JE, Reimann KA, Franchini G. 2005. Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus. Nat Med 11:740–747. doi: 10.1038/nm1261. [DOI] [PubMed] [Google Scholar]
41.Ollila J, Vihinen M. 2005. B cells. Int J Biochem Cell Biol 37:518–523. doi: 10.1016/j.biocel.2004.09.007. [DOI] [PubMed] [Google Scholar]
42.LeBien TW, Tedder TF. 2008. B lymphocytes: how they develop and function. Blood 112:1570–1580. doi: 10.1182/blood-2008-02-078071. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Monson NL. 2008. The natural history of B cells. Curr Opin Neurol 21(Suppl 1):S3–S8. doi: 10.1097/01.wco.0000313358.53553.c7. [DOI] [PubMed] [Google Scholar]
44.Byrd D, Amet T, Hu N, Lan J, Hu S, Yu Q. 2013. Primary human leukocyte subsets differentially express vaccinia virus receptors enriched in lipid rafts. J Virol 87:9301–9312. doi: 10.1128/JVI.01545-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Werden SJ, McFadden G. 2008. The role of cell signaling in poxvirus tropism: the case of the M-T5 host range protein of myxoma virus. Biochim Biophys Acta 1784:228–237. doi: 10.1016/j.bbapap.2007.08.001. [DOI] [PubMed] [Google Scholar]
46.Lalani AS, Masters J, Zeng W, Barrett J, Pannu R, Everett H, Arendt CW, McFadden G. 1999. Use of chemokine receptors by poxviruses. Science 286:1968–1971. doi: 10.1126/science.286.5446.1968. [DOI] [PubMed] [Google Scholar]
47.Byrd D, Shepherd N, Lan J, Hu N, Amet T, Yang K, Desai M, Yu Q. 2014. Primary human macrophages serve as vehicles for vaccinia virus replication and dissemination. J Virol 88:6819–6831. doi: 10.1128/JVI.03726-13. [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Tartaglia J, Perkus ME, Taylor J, Norton EK, Audonnet JC, Cox WI, Davis SW, van der Hoeven J, Meignier B, Riviere M. 1992. NYVAC: a highly attenuated strain of vaccinia virus. Virology 188:217–232. doi: 10.1016/0042-6822(92)90752-B. [DOI] [PubMed] [Google Scholar]
49.Taylor J, Meignier B, Tartaglia J, Languet B, VanderHoeven J, Franchini G, Trimarchi C, Paoletti E. 1995. Biological and immunogenic properties of a canarypox-rabies recombinant, ALVAC-RG (vCP65) in non-avian species. Vaccine 13:539–549. doi: 10.1016/0264-410X(94)00028-L. [DOI] [PubMed] [Google Scholar]
50.Engelmayer J, Larsson M, Subklewe M, Chahroudi A, Cox WI, Steinman RM, Bhardwaj N. 1999. Vaccinia virus inhibits the maturation of human dendritic cells: a novel mechanism of immune evasion. J Immunol 163:6762–6768. [PubMed] [Google Scholar]
51.Jenne L, Hauser C, Arrighi JF, Saurat JH, Hugin AW. 2000. Poxvirus as a vector to transduce human dendritic cells for immunotherapy: abortive infection but reduced APC function. Gene Ther 7:1575–1583. doi: 10.1038/sj.gt.3301287. [DOI] [PubMed] [Google Scholar]
52.Broder CC, Kennedy PE, Michaels F, Berger EA. 1994. Expression of foreign genes in cultured human primary macrophages using recombinant vaccinia virus vectors. Gene 142:167–174. doi: 10.1016/0378-1119(94)90257-7. [DOI] [PubMed] [Google Scholar]
53.Poland GA, Neff JM. 2003. Smallpox vaccine: problems and prospects. Immunol Allergy Clin North Am 23:731–743. doi: 10.1016/S0889-8561(03)00096-1. [DOI] [PubMed] [Google Scholar]
54.Czarnowicki T, Gonzalez J, Bonifacio KM, Shemer A, Xiangyu P, Kunjravia N, Malajian D, Fuentes-Duculan J, Esaki H, Noda S, Estrada Y, Xu H, Zheng X, Krueger JG, Guttman-Yassky E. 2016. Diverse activation and differentiation of multiple B-cell subsets in patients with atopic dermatitis but not in patients with psoriasis. J Allergy Clin Immunol 137:118–129. doi: 10.1016/j.jaci.2015.08.027. [DOI] [PubMed] [Google Scholar]
55.Grove J, Marsh M. 2011. The cell biology of receptor-mediated virus entry. J Cell Biol 195:1071–1082. doi: 10.1083/jcb.201108131. [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Foo CH, Lou H, Whitbeck JC, Ponce-de-León M, Atanasiu D, Eisenberg RJ, Cohen GH. 2009. Vaccinia virus L1 binds to cell surfaces and blocks virus entry independently of glycosaminoglycans. Virology 385:368–382. doi: 10.1016/j.virol.2008.12.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Lin CL, Chung CS, Heine HG, Chang W. 2000. Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo. J Virol 74:3353–3365. doi: 10.1128/jvi.74.7.3353-3365.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Chung CS, Hsiao JC, Chang YS, Chang W. 1998. A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate. J Virol 72:1577–1585. [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Bengali Z, Townsley AC, Moss B. 2009. Vaccinia virus strain differences in cell attachment and entry. Virology 389:132–140. doi: 10.1016/j.virol.2009.04.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Carter GC, Law M, Hollinshead M, Smith GL. 2005. Entry of the vaccinia virus intracellular mature virion and its interactions with glycosaminoglycans. J Gen Virol 86:1279–1290. doi: 10.1099/vir.0.80831-0. [DOI] [PubMed] [Google Scholar]
61.Marsh YV, Eppstein DA. 1987. Vaccinia virus and the EGF receptor: a portal for infectivity? J Cell Biochem 34:239–245. doi: 10.1002/jcb.240340403. [DOI] [PubMed] [Google Scholar]
62.Yang Z, Reynolds SE, Martens CA, Bruno DP, Porcella SF, Moss B. 2011. Expression profiling of the intermediate and late stages of poxvirus replication. J Virol 85:9899–9908. doi: 10.1128/JVI.05446-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
63.Yang Z, Maruri-Avidal L, Sisler J, Stuart CA, Moss B. 2013. Cascade regulation of vaccinia virus gene expression is modulated by multistage promoters. Virology 447:213–220. doi: 10.1016/j.virol.2013.09.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
64.Amanna IJ, Slifka MK, Crotty S. 2006. Immunity and immunological memory following smallpox vaccination. Immunol Rev 211:320–337. doi: 10.1111/j.0105-2896.2006.00392.x. [DOI] [PubMed] [Google Scholar]
65.Panchanathan V, Chaudhri G, Karupiah G. 2008. Correlates of protective immunity in poxvirus infection: where does antibody stand? Immunol Cell Biol 86:80–86. doi: 10.1038/sj.icb.7100118. [DOI] [PubMed] [Google Scholar]
66.Kennedy RB, Ovsyannikova IG, Jacobson RM, Poland GA. 2009. The immunology of smallpox vaccines. Curr Opin Immunol 21:314–320. doi: 10.1016/j.coi.2009.04.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
67.Moss B. 2011. Smallpox vaccines: targets of protective immunity. Immunol Rev 239:8–26. doi: 10.1111/j.1600-065X.2010.00975.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
68.Hammarlund E, Lewis MW, Hansen SG, Strelow LI, Nelson JA, Sexton GJ, Hanifin JM, Slifka MK. 2003. Duration of antiviral immunity after smallpox vaccination. Nat Med 9:1131–1137. doi: 10.1038/nm917. [DOI] [PubMed] [Google Scholar]
69.Kim JH, Excler JL, Michael NL. 2015. Lessons from the RV144 Thai phase III HIV-1 vaccine trial and the search for correlates of protection. Annu Rev Med 66:423–437. doi: 10.1146/annurev-med-052912-123749. [DOI] [PubMed] [Google Scholar]
70.Mackett M, Smith GL, Moss B. 1984. General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol 49:857–864. [DOI] [PMC free article] [PubMed] [Google Scholar]
71.Katsafanas GC, Moss B. 2007. Colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions. Cell Host Microbe 2:221–228. doi: 10.1016/j.chom.2007.08.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
72.Law M, Smith GL. 2004. Studying the binding and entry of the intracellular and extracellular enveloped forms of vaccinia virus. Methods Mol Biol 269:187–204. doi: 10.1385/1-59259-789-0:187. [DOI] [PubMed] [Google Scholar]
73.Doms RW, Blumenthal R, Moss B. 1990. Fusion of intra- and extracellular forms of vaccinia virus with the cell membrane. J Virol 64:4884–4892. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B1] 1.Blendon RJ, DesRoches CM, Benson JM, Herrmann MJ, Taylor-Clark K, Weldon KJ. 2003. The public and the smallpox threat. N Engl J Med 348:426–432. doi: 10.1056/NEJMsa023184. [DOI] [PubMed] [Google Scholar]

[B2] 2.Frey SE, Belshe RB. 2004. Poxvirus zoonoses–putting pocks into context. N Engl J Med 350:324–327. doi: 10.1056/NEJMp038208. [DOI] [PubMed] [Google Scholar]

[B3] 3.Essbauer S, Pfeffer M, Meyer H. 2010. Zoonotic poxviruses. Vet Microbiol 140:229–236. doi: 10.1016/j.vetmic.2009.08.026. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.Silva Gomes JA, de Araújo FF, de Souza Trindade G, Quinan BR, Drumond BP, Ferreira JMS, Mota BEF, Nogueira ML, Kroon EG, Santos Abrahão J, Côrrea-Oliveira R, da Fonseca FG. 2012. Immune modulation in primary vaccinia virus zoonotic human infections. Clin Dev Immunol 2012:974067. doi: 10.1155/2012/974067. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Sejvar JJ, Chowdary Y, Schomogyi M, Stevens J, Patel J, Karem K, Fischer M, Kuehnert MJ, Zaki SR, Paddock CD, Guarner J, Shieh WJ, Patton JL, Bernard N, Li Y, Olson VA, Kline RL, Loparev VN, Schmid DS, Beard B, Regnery RR, Damon IK. 2004. Human monkeypox infection: a family cluster in the midwestern United States. J Infect Dis 190:1833–1840. doi: 10.1086/425039. [DOI] [PubMed] [Google Scholar]

[B6] 6.Vorou RM, Papavassiliou VG, Pierroutsakos IN. 2008. Cowpox virus infection: an emerging health threat. Curr Opin Infect Dis 21:153–156. doi: 10.1097/QCO.0b013e3282f44c74. [DOI] [PubMed] [Google Scholar]

[B7] 7.Chan WM, McFadden G. 2014. Oncolytic poxviruses. Annu Rev Virol 1:119–141. doi: 10.1146/annurev-virology-031413-085442. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.Nichols AC, Yoo J, Um S, Mundi N, Palma DA, Fung K, Macneil SD, Koropatnick J, Mymryk JS, Barrett JW. 2014. Vaccinia virus outperforms a panel of other poxviruses as a potent oncolytic agent for the control of head and neck squamous cell carcinoma cell lines. Intervirology 57:17–22. doi: 10.1159/000353854. [DOI] [PubMed] [Google Scholar]

[B9] 9.Mejías-Pérez E, Carreño-Fuentes L, Esteban M. 2018. Development of a safe and effective vaccinia virus oncolytic vector WR-Delta4 with a set of gene deletions on several viral pathways. Mol Ther Oncolytics 8:27–40. doi: 10.1016/j.omto.2017.12.002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Amara RR, Villinger F, Altman JD, Lydy SL, O'Neil SP, Staprans SI, Montefiori DC, Xu Y, Herndon JG, Wyatt LS, Candido MA, Kozyr NL, Earl PL, Smith JM, Ma HL, Grimm BD, Hulsey ML, Miller J, McClure HM, McNicholl JM, Moss B, Robinson HL. 2001. Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292:69–74. doi: 10.1126/science.1058915. [DOI] [PubMed] [Google Scholar]

[B11] 11.Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C, de Souza M, Adams E, Benenson M, Gurunathan S, Tartaglia J, McNeil JG, Francis DP, Stablein D, Birx DL, Chunsuttiwat S, Khamboonruang C, Thongcharoen P, Robb ML, Michael NL, Kunasol P, Kim JH, MOPH-TAVEG Investigators. 2009. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 361:2209–2220. doi: 10.1056/NEJMoa0908492. [DOI] [PubMed] [Google Scholar]

[B12] 12.Weiss S, Yitzhaki S, Shapira SC. 2015. Lessons to be learned from recent biosafety incidents in the United States. Isr Med Assoc J 17:269–273. [PubMed] [Google Scholar]

[B13] 13.Falendysz EA, Lopera JG, Doty JB, Nakazawa Y, Crill C, Lorenzsonn F, Kalemba LN, Ronderos MD, Mejia A, Malekani JM, Karem K, Carroll DS, Osorio JE, Rocke TE. 2017. Characterization of monkeypox virus infection in African rope squirrels (Funisciurus sp.). PLoS Negl Trop Dis 11:e0005809. doi: 10.1371/journal.pntd.0005809. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Chen N, Li G, Liszewski MK, Atkinson JP, Jahrling PB, Feng Z, Schriewer J, Buck C, Wang C, Lefkowitz EJ, Esposito JJ, Harms T, Damon IK, Roper RL, Upton C, Buller RM. 2005. Virulence differences between monkeypox virus isolates from West Africa and the Congo basin. Virology 340:46–63. doi: 10.1016/j.virol.2005.05.030. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Damaso CR, Esposito JJ, Condit RC, Moussatche N. 2000. An emergent poxvirus from humans and cattle in Rio de Janeiro State: Cantagalo virus may derive from Brazilian smallpox vaccine. Virology 277:439–449. doi: 10.1006/viro.2000.0603. [DOI] [PubMed] [Google Scholar]

[B16] 16.Dhar AD, Werchniak AE, Li Y, Brennick JB, Goldsmith CS, Kline R, Damon I, Klaus SN. 2004. Tanapox infection in a college student. N Engl J Med 350:361–366. doi: 10.1056/NEJMoa031467. [DOI] [PubMed] [Google Scholar]

[B17] 17.Stich A, Meyer H, Kohler B, Fleischer K. 2002. Tanapox: first report in a European traveller and identification by PCR. Trans R Soc Trop Med Hyg 96:178–179. doi: 10.1016/s0035-9203(02)90295-6. [DOI] [PubMed] [Google Scholar]

[B18] 18.Kolhapure RM, Deolankar RP, Tupe CD, Raut CG, Basu A, Dama BM, Pawar SD, Joshi MV, Padbidri VS, Goverdhan MK, Banerjee K. 1997. Investigation of buffalopox outbreaks in Maharashtra State during 1992-1996. Indian J Med Res 106:441–446. [PubMed] [Google Scholar]

[B19] 19.Gerdts V, Mutwiri GK, Tikoo SK, Babiuk LA. 2006. Mucosal delivery of vaccines in domestic animals. Vet Res 37:487–510. doi: 10.1051/vetres:2006012. [DOI] [PubMed] [Google Scholar]

[B20] 20.Yamada T, Hamano Y, Hasegawa N, Seo E, Fukuda K, Yokoyama KK, Hyodo I, Abei M. 2018. Oncolytic virotherapy and gene therapy strategies for hepatobiliary cancers. Curr Cancer Drug Targets 18:188–201. doi: 10.2174/1568009617666170330123841. [DOI] [PubMed] [Google Scholar]

[B21] 21.Park SH, Breitbach CJ, Lee J, Park JO, Lim HY, Kang WK, Moon A, Mun JH, Sommermann EM, Maruri Avidal L, Patt R, Pelusio A, Burke J, Hwang TH, Kirn D, Park YS. 2015. Phase 1b trial of biweekly intravenous Pexa-Vec (JX-594), an oncolytic and immunotherapeutic vaccinia virus in colorectal cancer. Mol Ther 23:1532–1540. doi: 10.1038/mt.2015.109. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B22] 22.Gómez CE, Nájera JL, Perdiguero B, García-Arriaza J, Sorzano COS, Jiménez V, González-Sanz R, Jiménez JL, Muñoz-Fernández MA, López Bernaldo de Quirós JC, Guardo AC, García F, Gatell JM, Plana M, Esteban M. 2011. The HIV/AIDS vaccine candidate MVA-B administered as a single immunogen in humans triggers robust, polyfunctional, and selective effector memory T cell responses to HIV-1 antigens. J Virol 85:11468–11478. doi: 10.1128/JVI.05165-11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23.Cavenaugh JS, Awi D, Mendy M, Hill AV, Whittle H, McConkey SJ. 2011. Partially randomized, non-blinded trial of DNA and MVA therapeutic vaccines based on hepatitis B virus surface protein for chronic HBV infection. PLoS One 6:e14626. doi: 10.1371/journal.pone.0014626. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Berthoud TK, Hamill M, Lillie PJ, Hwenda L, Collins KA, Ewer KJ, Milicic A, Poyntz HC, Lambe T, Fletcher HA, Hill AV, Gilbert SC. 2011. Potent CD8+ T-cell immunogenicity in humans of a novel heterosubtypic influenza A vaccine, MVA-NP+M1. Clin Infect Dis 52:1–7. doi: 10.1093/cid/ciq015. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25.Porter DW, Thompson FM, Berthoud TK, Hutchings CL, Andrews L, Biswas S, Poulton I, Prieur E, Correa S, Rowland R, Lang T, Williams J, Gilbert SC, Sinden RE, Todryk S, Hill AV. 2011. A human Phase I/IIa malaria challenge trial of a polyprotein malaria vaccine. Vaccine 29:7514–7522. doi: 10.1016/j.vaccine.2011.03.083. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26.Tameris MD, Hatherill M, Landry BS, Scriba TJ, Snowden MA, Lockhart S, Shea JE, McClain JB, Hussey GD, Hanekom WA, Mahomed H, McShane H. 2013. Safety and efficacy of MVA85A, a new tuberculosis vaccine, in infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. Lancet 381:1021–1028. doi: 10.1016/S0140-6736(13)60177-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27.Rahal A, Musher B. 2017. Oncolytic viral therapy for pancreatic cancer. J Surg Oncol 116:94–103. doi: 10.1002/jso.24626. [DOI] [PubMed] [Google Scholar]

[B28] 28.Jebar AH, Errington-Mais F, Vile RG, Selby PJ, Melcher AA, Griffin S. 2015. Progress in clinical oncolytic virus-based therapy for hepatocellular carcinoma. J Gen Virol 96:1533–1550. doi: 10.1099/vir.0.000098. [DOI] [PubMed] [Google Scholar]

[B29] 29.Downs-Canner S, Guo ZS, Ravindranathan R, Breitbach CJ, O'Malley ME, Jones HL, Moon A, McCart JA, Shuai Y, Zeh HJ, Bartlett DL. 2016. Phase 1 study of intravenous oncolytic poxvirus (vvDD) in patients with advanced solid cancers. Mol Ther 24:1492–1501. doi: 10.1038/mt.2016.101. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30.Ura T, Okuda K, Shimada M. 2014. Developments in viral vector-based vaccines. Vaccines 2:624–641. doi: 10.3390/vaccines2030624. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B31] 31.Moss B. 1996. Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety. Proc Natl Acad Sci U S A 93:11341–11348. doi: 10.1073/pnas.93.21.11341. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32.McFadden G. 2005. Poxvirus tropism. Nat Rev Microbiol 3:201–213. doi: 10.1038/nrmicro1099. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B33] 33.Wein LM, Wu JT, Kirn DH. 2003. Validation and analysis of a mathematical model of a replication-competent oncolytic virus for cancer treatment: implications for virus design and delivery. Cancer Res 63:1317–1324. [PubMed] [Google Scholar]

[B34] 34.Carroll MW, Moss B. 1997. Poxviruses as expression vectors. Curr Opin Biotechnol 8:573–577. doi: 10.1016/S0958-1669(97)80031-6. [DOI] [PubMed] [Google Scholar]

[B35] 35.Smith GL, Moss B. 1983. Infectious poxvirus vectors have capacity for at least 25,000 base pairs of foreign DNA. Gene 25:21–28. doi: 10.1016/0378-1119(83)90163-4. [DOI] [PubMed] [Google Scholar]

[B36] 36.Yu Q, Jones B, Hu N, Chang H, Ahmad S, Liu J, Parrington M, Ostrowski M. 2006. Comparative analysis of tropism between canarypox (ALVAC) and vaccinia viruses reveals a more restricted and preferential tropism of ALVAC for human cells of the monocytic lineage. Vaccine 24:6376–6391. doi: 10.1016/j.vaccine.2006.06.011. [DOI] [PubMed] [Google Scholar]

[B37] 37.Hu N, Yu R, Shikuma C, Shiramizu B, Ostrwoski MA, Yu Q. 2009. Role of cell signaling in poxvirus-mediated foreign gene expression in mammalian cells. Vaccine 27:2994–3006. doi: 10.1016/j.vaccine.2009.02.103. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B38] 38.Sanchez-Puig JM, Sanchez L, Roy G, Blasco R. 2004. Susceptibility of different leukocyte cell types to vaccinia virus infection. Virol J 1:10. doi: 10.1186/1743-422X-1-10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B39] 39.Chahroudi A, Chavan R, Kozyr N, Koyzr N, Waller EK, Silvestri G, Feinberg MB. 2005. Vaccinia virus tropism for primary hematolymphoid cells is determined by restricted expression of a unique virus receptor. J Virol 79:10397–10407. doi: 10.1128/JVI.79.16.10397-10407.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B40] 40.Edghill-Smith Y, Golding H, Manischewitz J, King LR, Scott D, Bray M, Nalca A, Hooper JW, Whitehouse CA, Schmitz JE, Reimann KA, Franchini G. 2005. Smallpox vaccine-induced antibodies are necessary and sufficient for protection against monkeypox virus. Nat Med 11:740–747. doi: 10.1038/nm1261. [DOI] [PubMed] [Google Scholar]

[B41] 41.Ollila J, Vihinen M. 2005. B cells. Int J Biochem Cell Biol 37:518–523. doi: 10.1016/j.biocel.2004.09.007. [DOI] [PubMed] [Google Scholar]

[B42] 42.LeBien TW, Tedder TF. 2008. B lymphocytes: how they develop and function. Blood 112:1570–1580. doi: 10.1182/blood-2008-02-078071. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B43] 43.Monson NL. 2008. The natural history of B cells. Curr Opin Neurol 21(Suppl 1):S3–S8. doi: 10.1097/01.wco.0000313358.53553.c7. [DOI] [PubMed] [Google Scholar]

[B44] 44.Byrd D, Amet T, Hu N, Lan J, Hu S, Yu Q. 2013. Primary human leukocyte subsets differentially express vaccinia virus receptors enriched in lipid rafts. J Virol 87:9301–9312. doi: 10.1128/JVI.01545-13. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B45] 45.Werden SJ, McFadden G. 2008. The role of cell signaling in poxvirus tropism: the case of the M-T5 host range protein of myxoma virus. Biochim Biophys Acta 1784:228–237. doi: 10.1016/j.bbapap.2007.08.001. [DOI] [PubMed] [Google Scholar]

[B46] 46.Lalani AS, Masters J, Zeng W, Barrett J, Pannu R, Everett H, Arendt CW, McFadden G. 1999. Use of chemokine receptors by poxviruses. Science 286:1968–1971. doi: 10.1126/science.286.5446.1968. [DOI] [PubMed] [Google Scholar]

[B47] 47.Byrd D, Shepherd N, Lan J, Hu N, Amet T, Yang K, Desai M, Yu Q. 2014. Primary human macrophages serve as vehicles for vaccinia virus replication and dissemination. J Virol 88:6819–6831. doi: 10.1128/JVI.03726-13. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B48] 48.Tartaglia J, Perkus ME, Taylor J, Norton EK, Audonnet JC, Cox WI, Davis SW, van der Hoeven J, Meignier B, Riviere M. 1992. NYVAC: a highly attenuated strain of vaccinia virus. Virology 188:217–232. doi: 10.1016/0042-6822(92)90752-B. [DOI] [PubMed] [Google Scholar]

[B49] 49.Taylor J, Meignier B, Tartaglia J, Languet B, VanderHoeven J, Franchini G, Trimarchi C, Paoletti E. 1995. Biological and immunogenic properties of a canarypox-rabies recombinant, ALVAC-RG (vCP65) in non-avian species. Vaccine 13:539–549. doi: 10.1016/0264-410X(94)00028-L. [DOI] [PubMed] [Google Scholar]

[B50] 50.Engelmayer J, Larsson M, Subklewe M, Chahroudi A, Cox WI, Steinman RM, Bhardwaj N. 1999. Vaccinia virus inhibits the maturation of human dendritic cells: a novel mechanism of immune evasion. J Immunol 163:6762–6768. [PubMed] [Google Scholar]

[B51] 51.Jenne L, Hauser C, Arrighi JF, Saurat JH, Hugin AW. 2000. Poxvirus as a vector to transduce human dendritic cells for immunotherapy: abortive infection but reduced APC function. Gene Ther 7:1575–1583. doi: 10.1038/sj.gt.3301287. [DOI] [PubMed] [Google Scholar]

[B52] 52.Broder CC, Kennedy PE, Michaels F, Berger EA. 1994. Expression of foreign genes in cultured human primary macrophages using recombinant vaccinia virus vectors. Gene 142:167–174. doi: 10.1016/0378-1119(94)90257-7. [DOI] [PubMed] [Google Scholar]

[B53] 53.Poland GA, Neff JM. 2003. Smallpox vaccine: problems and prospects. Immunol Allergy Clin North Am 23:731–743. doi: 10.1016/S0889-8561(03)00096-1. [DOI] [PubMed] [Google Scholar]

[B54] 54.Czarnowicki T, Gonzalez J, Bonifacio KM, Shemer A, Xiangyu P, Kunjravia N, Malajian D, Fuentes-Duculan J, Esaki H, Noda S, Estrada Y, Xu H, Zheng X, Krueger JG, Guttman-Yassky E. 2016. Diverse activation and differentiation of multiple B-cell subsets in patients with atopic dermatitis but not in patients with psoriasis. J Allergy Clin Immunol 137:118–129. doi: 10.1016/j.jaci.2015.08.027. [DOI] [PubMed] [Google Scholar]

[B55] 55.Grove J, Marsh M. 2011. The cell biology of receptor-mediated virus entry. J Cell Biol 195:1071–1082. doi: 10.1083/jcb.201108131. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B56] 56.Foo CH, Lou H, Whitbeck JC, Ponce-de-León M, Atanasiu D, Eisenberg RJ, Cohen GH. 2009. Vaccinia virus L1 binds to cell surfaces and blocks virus entry independently of glycosaminoglycans. Virology 385:368–382. doi: 10.1016/j.virol.2008.12.019. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B57] 57.Lin CL, Chung CS, Heine HG, Chang W. 2000. Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo. J Virol 74:3353–3365. doi: 10.1128/jvi.74.7.3353-3365.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B58] 58.Chung CS, Hsiao JC, Chang YS, Chang W. 1998. A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate. J Virol 72:1577–1585. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B59] 59.Bengali Z, Townsley AC, Moss B. 2009. Vaccinia virus strain differences in cell attachment and entry. Virology 389:132–140. doi: 10.1016/j.virol.2009.04.012. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B60] 60.Carter GC, Law M, Hollinshead M, Smith GL. 2005. Entry of the vaccinia virus intracellular mature virion and its interactions with glycosaminoglycans. J Gen Virol 86:1279–1290. doi: 10.1099/vir.0.80831-0. [DOI] [PubMed] [Google Scholar]

[B61] 61.Marsh YV, Eppstein DA. 1987. Vaccinia virus and the EGF receptor: a portal for infectivity? J Cell Biochem 34:239–245. doi: 10.1002/jcb.240340403. [DOI] [PubMed] [Google Scholar]

[B62] 62.Yang Z, Reynolds SE, Martens CA, Bruno DP, Porcella SF, Moss B. 2011. Expression profiling of the intermediate and late stages of poxvirus replication. J Virol 85:9899–9908. doi: 10.1128/JVI.05446-11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B63] 63.Yang Z, Maruri-Avidal L, Sisler J, Stuart CA, Moss B. 2013. Cascade regulation of vaccinia virus gene expression is modulated by multistage promoters. Virology 447:213–220. doi: 10.1016/j.virol.2013.09.007. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B64] 64.Amanna IJ, Slifka MK, Crotty S. 2006. Immunity and immunological memory following smallpox vaccination. Immunol Rev 211:320–337. doi: 10.1111/j.0105-2896.2006.00392.x. [DOI] [PubMed] [Google Scholar]

[B65] 65.Panchanathan V, Chaudhri G, Karupiah G. 2008. Correlates of protective immunity in poxvirus infection: where does antibody stand? Immunol Cell Biol 86:80–86. doi: 10.1038/sj.icb.7100118. [DOI] [PubMed] [Google Scholar]

[B66] 66.Kennedy RB, Ovsyannikova IG, Jacobson RM, Poland GA. 2009. The immunology of smallpox vaccines. Curr Opin Immunol 21:314–320. doi: 10.1016/j.coi.2009.04.004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B67] 67.Moss B. 2011. Smallpox vaccines: targets of protective immunity. Immunol Rev 239:8–26. doi: 10.1111/j.1600-065X.2010.00975.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B68] 68.Hammarlund E, Lewis MW, Hansen SG, Strelow LI, Nelson JA, Sexton GJ, Hanifin JM, Slifka MK. 2003. Duration of antiviral immunity after smallpox vaccination. Nat Med 9:1131–1137. doi: 10.1038/nm917. [DOI] [PubMed] [Google Scholar]

[B69] 69.Kim JH, Excler JL, Michael NL. 2015. Lessons from the RV144 Thai phase III HIV-1 vaccine trial and the search for correlates of protection. Annu Rev Med 66:423–437. doi: 10.1146/annurev-med-052912-123749. [DOI] [PubMed] [Google Scholar]

[B70] 70.Mackett M, Smith GL, Moss B. 1984. General method for production and selection of infectious vaccinia virus recombinants expressing foreign genes. J Virol 49:857–864. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B71] 71.Katsafanas GC, Moss B. 2007. Colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions. Cell Host Microbe 2:221–228. doi: 10.1016/j.chom.2007.08.005. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B72] 72.Law M, Smith GL. 2004. Studying the binding and entry of the intracellular and extracellular enveloped forms of vaccinia virus. Methods Mol Biol 269:187–204. doi: 10.1385/1-59259-789-0:187. [DOI] [PubMed] [Google Scholar]

[B73] 73.Doms RW, Blumenthal R, Moss B. 1990. Fusion of intra- and extracellular forms of vaccinia virus with the cell membrane. J Virol 64:4884–4892. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Primary Human B Cells at Different Differentiation and Maturation Stages Exhibit Distinct Susceptibilities to Vaccinia Virus Binding and Infection

Nicole Shepherd

Jie Lan

Wei Li

Sushmita Rane

Qigui Yu

Roles

ABSTRACT

INTRODUCTION

RESULTS

VACV robustly bound to but moderately or weakly infected primary human B cells.

FIG 1.

VACV aggressively bound to multiple subsets of B cells but not plasmablasts.

FIG 2.

VACV effectively infected subpopulations of memory B cells.

FIG 3.

VACV underwent an abortive infection in unstimulated primary human B cells at the late stage of VACV gene expression.

FIG 4.

VACV underwent a productive infection in activated primary human B cells.

FIG 5.

TABLE 1.

DISCUSSION

MATERIALS AND METHODS

Ethics statement.

Preparation and activation of primary human B cells.

Antibodies and FCM.

TABLE 2.

VACV preparation, binding, and infection.

Confocal microscopy analysis of VACV binding.

RT-PCR analysis of VACV gene expression.

Multiplex immunoassays and ELISAs.

Statistical analysis.

ACKNOWLEDGMENTS

REFERENCES

ACTIONS

PERMALINK

RESOURCES

Cite

Add to Collections

PERMALINK

Primary Human B Cells at Different Differentiation and Maturation Stages Exhibit Distinct Susceptibilities to Vaccinia Virus Binding and Infection

Nicole Shepherd

Jie Lan

Wei Li

Sushmita Rane

Qigui Yu

Roles

ABSTRACT

INTRODUCTION

RESULTS

VACV robustly bound to but moderately or weakly infected primary human B cells.

FIG 1.

VACV aggressively bound to multiple subsets of B cells but not plasmablasts.

FIG 2.

VACV effectively infected subpopulations of memory B cells.

FIG 3.

VACV underwent an abortive infection in unstimulated primary human B cells at the late stage of VACV gene expression.

FIG 4.

VACV underwent a productive infection in activated primary human B cells.

FIG 5.

TABLE 1.

DISCUSSION

MATERIALS AND METHODS

Ethics statement.

Preparation and activation of primary human B cells.

Antibodies and FCM.

TABLE 2.

VACV preparation, binding, and infection.

Confocal microscopy analysis of VACV binding.

RT-PCR analysis of VACV gene expression.

Multiplex immunoassays and ELISAs.

Statistical analysis.

ACKNOWLEDGMENTS

REFERENCES

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases