Late-stage MC38 tumours recapitulate features of human colorectal cancer - implications for appropriate timepoint selection in preclinical studies.

Anti-tumour T cell responses play a crucial role in controlling the progression of colorectal cancer (CRC), making this disease a promising candidate for immunotherapy. However, responses to immune-targeted therapies are currently limited to subpopulations of patients and specific types of cancer. Clinical studies have therefore focussed on identifying biomarkers that predict immunotherapy responses and elucidating the immunological landscapes of different cancers. Meanwhile, our understanding of how preclinical tumour models resemble human disease has fallen behind, despite their crucial role in immune-targeted drug development. A deeper understanding of these models is therefore needed to improve the development of immunotherapies and the translation of findings made in these systems. MC38 colon adenocarcinoma is a widely used preclinical model, yet how it recapitulates human colorectal cancer remains poorly defined. This study investigated the tumour-T cell immune landscape of MC38 tumours using histology, immunohistochemistry, and flow cytometry. We demonstrate that early-stage tumours exhibit a nascent TME, lacking important immune-resistance mechanisms of clinical interest, while late-stage tumours exhibit a mature TME resembling human tumours, with desmoplasia, T cell exhaustion, and T cell exclusion. Consequently, these findings clarify appropriate timepoint selection in the MC38 model when investigating both immunotherapies and mechanisms that contribute to immunotherapy resistance. Overall, this study provides a valuable resource that will enable appropriate application of the MC38 model and expedite the development and clinical translation of new immunotherapies.

Mucosal TLR2-activating protein-based vaccination induces potent pulmonary immunity and protection against SARS-CoV-2 in mice.

Current vaccines against SARS-CoV-2 substantially reduce mortality, but protection against infection is less effective. Enhancing immunity in the respiratory tract, via mucosal vaccination, may provide protection against infection and minimise viral spread. Here, we report testing of a subunit vaccine in mice, consisting of SARS-CoV-2 Spike protein with a TLR2-stimulating adjuvant (Pam2Cys), delivered to mice parenterally or mucosally. Both routes of vaccination induce substantial neutralising antibody (nAb) titres, however, mucosal vaccination uniquely generates anti-Spike IgA, increases nAb in the serum and airways, and increases lung CD4+ T-cell responses. TLR2 is expressed by respiratory epithelia and immune cells. Using TLR2 deficient chimeric mice, we determine that TLR2 expression in either compartment facilitates early innate responses to mucosal vaccination. By contrast, TLR2 on hematopoietic cells is essential for optimal lung-localised, antigen-specific responses. In K18-hACE2 mice, vaccination provides complete protection against disease and sterilising lung immunity against SARS-CoV-2, with a short-term non-specific protective effect from mucosal Pam2Cys alone. These data support mucosal vaccination as a strategy to improve protection in the respiratory tract against SARS-CoV-2 and other respiratory viruses.

Functional paralysis of human natural killer cells by alphaherpesviruses.

Natural killer (NK) cells are implicated as important anti-viral immune effectors in varicella zoster virus (VZV) infection. VZV can productively infect human NK cells, yet it is unknown how, or if, VZV can directly affect NK cell function. Here we demonstrate that VZV potently impairs the ability of NK cells to respond to target cell stimulation in vitro, leading to a loss of both cytotoxic and cytokine responses. Remarkably, not only were VZV infected NK cells affected, but VZV antigen negative NK cells that were exposed to virus in culture were also inhibited. This powerful impairment of function was dependent on direct contact between NK cells and VZV infected inoculum cells. Profiling of the NK cell surface receptor phenotype by multiparameter flow cytometry revealed that functional receptor expression is predominantly stable. Furthermore, inhibited NK cells were still capable of releasing cytotoxic granules when the stimulation signal bypassed receptor/ligand interactions and early signalling, suggesting that VZV paralyses NK cells from responding. Phosflow examination of key components in the degranulation signalling cascade also demonstrated perturbation following culture with VZV. In addition to inhibiting degranulation, IFN-γ and TNF production were also repressed by VZV co-culture, which was most strongly regulated in VZV infected NK cells. Interestingly, the closely related virus, herpes simplex virus type 1 (HSV-1), was also capable of efficiently infecting NK cells in a cell-associated manner, and demonstrated a similar capacity to render NK cells unresponsive to target cell stimulation-however HSV-1 differentially targeted cytokine production compared to VZV. Our findings progress a growing understanding of pathogen inhibition of NK cell function, and reveal a previously unreported strategy for VZV to manipulate the immune response.

Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies.

The murine cytomegalovirus protein M45 protects infected mouse cells from necroptotic death and, when heterologously expressed, can protect human cells from necroptosis induced by tumour necrosis factor receptor (TNFR) activation. Here, we show that the N-terminal 90 residues of the M45 protein, which contain a RIP homotypic interaction motif (RHIM), are sufficient to confer protection against TNFR-induced necroptosis. This N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils and interacts with the RHIMs of the human kinases RIPK1 and RIPK3, and the Z-DNA binding protein 1 (ZBP1), to form heteromeric amyloid fibrils in vitro Mutation of the tetrad residues in the M45 RHIM attenuates homo- and hetero-amyloid assembly by M45, suggesting that the amyloidogenic nature of the M45 RHIM underlies its biological activity. The M45 RHIM preferentially interacts with RIPK3 and ZBP1 over RIPK1 and alters the properties of the host RHIM protein assemblies. Our results indicate that M45 mimics the interactions made by RIPK1 or ZBP1 with RIPK3, thereby forming heteromeric amyloid structures, which may explain its ability to inhibit necroptosis.

Infection and Functional Modulation of Human Monocytes and Macrophages by Varicella-Zoster Virus.

Varicella-zoster virus (VZV) is associated with viremia during primary infection that is presumed to stem from infection of circulating immune cells. While VZV has been shown to be capable of infecting a number of different subsets of circulating immune cells, such as T cells, dendritic cells, and NK cells, less is known about the interaction between VZV and monocytes. Here, we demonstrate that blood-derived human monocytes are permissive to VZV replication in vitro VZV-infected monocytes exhibited each temporal class of VZV gene expression, as evidenced by immunofluorescent staining. VZV virions were observed on the cell surface and viral nucleocapsids were observed in the nucleus of VZV-infected monocytes by scanning electron microscopy. In addition, VZV-infected monocytes were able to transfer infectious virus to human fibroblasts. Infected monocytes displayed impaired dextran-mediated endocytosis, and cell surface immunophenotyping revealed the downregulation of CD14, HLA-DR, CD11b, and the macrophage colony-stimulating factor (M-CSF) receptor. Analysis of the impact of VZV infection on M-CSF-stimulated monocyte-to-macrophage differentiation demonstrated the loss of cell viability, indicating that VZV-infected monocytes were unable to differentiate into viable macrophages. In contrast, macrophages differentiated from monocytes prior to exposure to VZV were highly permissive to infection. This study defines the permissiveness of these myeloid cell types to productive VZV infection and identifies the functional impairment of VZV-infected monocytes.IMPORTANCE Primary VZV infection results in the widespread dissemination of the virus throughout the host. Viral transportation is known to be directly influenced by susceptible immune cells in the circulation. Moreover, infection of immune cells by VZV results in attenuation of the antiviral mechanisms used to control infection and limit spread. Here, we provide evidence that human monocytes, which are highly abundant in the circulation, are permissive to productive VZV infection. Furthermore, monocyte-derived macrophages were also highly permissive to VZV infection, although VZV-infected monocytes were unable to differentiate into macrophages. Exploring the relationships between VZV and permissive immune cells, such as human monocytes and macrophages, elucidates novel immune evasion strategies and provides further insight into the control that VZV has over the immune system.

Varicella-Zoster Virus ORF63 Protects Human Neuronal and Keratinocyte Cell Lines from Apoptosis and Changes Its Localization upon Apoptosis Induction.

There are many facets of varicella-zoster virus (VZV) pathogenesis that are not fully understood, such as the mechanisms involved in the establishment of lifelong latency, reactivation, and development of serious conditions like postherpetic neuralgia (PHN). Virus-encoded modulation of apoptosis has been suggested to play an important role in these processes. VZV open reading frame 63 (ORF63) has been shown to modulate apoptosis in a cell-type-specific manner, but the impact of ORF63 on cell death pathways has not been examined in isolation in the context of human cells. We sought to elucidate the effect of VZV ORF63 on apoptosis induction in human neuron and keratinocyte cell lines. VZV ORF63 was shown to protect differentiated SH-SY5Y neuronal cells against staurosporine-induced apoptosis. In addition, VZV infection did not induce high levels of apoptosis in the HaCaT human keratinocyte line, highlighting a delay in apoptosis induction. VZV ORF63 was shown to protect HaCaT cells against both staurosporine- and Fas ligand-induced apoptosis. Confocal microscopy was utilized to examine VZV ORF63 localization during apoptosis induction. In VZV infection and ORF63 expression alone, VZV ORF63 became more cytoplasmic, with aggregate formation during apoptosis induction. Taken together, this suggests that VZV ORF63 protects both differentiated SH-SY5Y cells and HaCaT cells from apoptosis induction and may mediate this effect through its localization change during apoptosis. VZV ORF63 is a prominent VZV gene product in both productive and latent infection and thus may play a critical role in VZV pathogenesis by aiding neuron and keratinocyte survival.IMPORTANCE VZV, a human-specific alphaherpesvirus, causes chicken pox during primary infection and establishes lifelong latency in the dorsal root ganglia (DRG). Reactivation of VZV causes shingles, which is often followed by a prolonged pain syndrome called postherpetic neuralgia. It has been suggested that the ability of the virus to modulate cell death pathways is linked to its ability to establish latency and reactivate. The significance of our research lies in investigating the ability of ORF63, a VZV gene product, to inhibit apoptosis in novel cell types crucial for VZV pathogenesis. This will allow an increased understanding of critical enigmatic components of VZV pathogenesis.

Human Cytomegalovirus-Encoded Human Interleukin-10 (IL-10) Homolog Amplifies Its Immunomodulatory Potential by Upregulating Human IL-10 in Monocytes.

UNLABELLED: The human cytomegalovirus (HCMV) gene UL111A encodes cytomegalovirus-encoded human interleukin-10 (cmvIL-10), a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). This viral homolog exhibits a range of immunomodulatory functions, including suppression of proinflammatory cytokine production and dendritic cell (DC) maturation, as well as inhibition of major histocompatibility complex (MHC) class I and class II. Here, we present data showing that cmvIL-10 upregulates hIL-10, and we identify CD14(+)monocytes and monocyte-derived macrophages and DCs as major sources of hIL-10 secretion in response to cmvIL-10. Monocyte activation was not a prerequisite for cmvIL-10-mediated upregulation of hIL-10, which was dose dependent and controlled at the transcriptional level. Furthermore, cmvIL-10 upregulated expression of tumor progression locus 2 (TPL2), which is a regulator of the positive hIL-10 feedback loop, whereas expression of a negative regulator of the hIL-10 feedback loop, dual-specificity phosphatase 1 (DUSP1), remained unchanged. Engagement of the hIL-10 receptor (hIL-10R) by cmvIL-10 led to upregulation of heme oxygenase 1 (HO-1), an enzyme linked with suppression of inflammatory responses, and this upregulation was required for cmvIL-10-mediated upregulation of hIL-10. We also demonstrate an important role for both phosphatidylinositol 3-kinase (PI3K) and STAT3 in the upregulation of HO-1 and hIL-10 by cmvIL-10. In addition to upregulating hIL-10, cmvIL-10 could exert a direct immunomodulatory function, as demonstrated by its capacity to upregulate expression of cell surface CD163 when hIL-10 was neutralized. This study identifies a mechanistic basis for cmvIL-10 function, including the capacity of this viral cytokine to potentially amplify its immunosuppressive impact by upregulating hIL-10 expression. IMPORTANCE: Human cytomegalovirus (HCMV) is a large, double-stranded DNA virus that causes significant human disease, particularly in the congenital setting and in solid-organ and hematopoietic stem cell transplant patients. A prominent feature of HCMV is the wide range of viral gene products that it encodes which function to modulate host defenses. One of these is cmvIL-10, which is a homolog of the potent immunomodulatory cytokine human interleukin 10 (hIL-10). In this study, we report that, in addition to exerting a direct biological impact, cmvIL-10 upregulates the expression of hIL-10 by primary blood-derived monocytes and that it does so by modulating existing cellular pathways. This capacity of cmvIL-10 to upregulate hIL-10 represents a mechanism by which HCMV may amplify its immunomodulatory impact during infection.

Varicella-zoster virus glycoprotein I is essential for spread in dorsal root ganglia and facilitates axonal localization of structural virion components in neuronal cultures.

Neurons of the sensory ganglia are the major site of varicella-zoster virus (VZV) latency and may undergo productive infection during reactivation. Although the VZV glycoprotein E/glycoprotein I (gE/gI) complex is known to be critical for neurovirulence, few studies have assessed the roles of these proteins during infection of dorsal root ganglia (DRG) due to the high human specificity of the virus. Here, we show that the VZV glycoprotein I gene is an important neurotropic gene responsible for mediating the spread of virus in neuronal cultures and explanted DRG. Inoculation of differentiated SH-SY5Y neuronal cell cultures with a VZV gI gene deletion strain (VZV rOkaΔgI) showed a large reduction in the percentage of cells infected and significantly smaller plaque sizes in a comparison with cultures infected with the parental strain (VZV rOka). In contrast, VZV rOkaΔgI was not significantly attenuated in fibroblast cultures, demonstrating a cell type-specific role for VZV gI. Analysis of rOkaΔgI protein localization by immunofluorescent staining revealed aberrant localization of viral glycoprotein and capsid proteins, with little or no staining present in the axons of differentiated SH-SY5Y cells infected with rOkaΔgI, yet axonal vesicle trafficking was not impaired. Further studies utilizing explanted human DRG indicated that VZV gI is required for the spread of virus within DRG. These data demonstrate a role for VZV gI in the cell-to-cell spread of virus during productive replication in neuronal cells and a role in facilitating the access of virion components to axons.

Human cytomegalovirus interleukin-10 polarizes monocytes toward a deactivated M2c phenotype to repress host immune responses.

Several human cytomegalovirus (HCMV) genes encode products that modulate cellular functions in a manner likely to enhance viral pathogenesis. This includes UL111A, which encodes homologs of human interleukin-10 (hIL-10). Depending upon signals received, monocytes and macrophages become polarized to either classically activated (M1 proinflammatory) or alternatively activated (M2 anti-inflammatory) subsets. Skewing of polarization toward an M2 subset may benefit the virus by limiting the proinflammatory responses to infection, and so we determined whether HCMV-encoded viral IL-10 influenced monocyte polarization. Recombinant viral IL-10 protein polarized CD14(+) monocytes toward an anti-inflammatory M2 subset with an M2c phenotype, as demonstrated by high expression of CD163 and CD14 and suppression of major histocompatibility complex (MHC) class II. Significantly, in the context of productive HCMV infection, viral IL-10 produced by infected cells polarized uninfected monocytes toward an M2c phenotype. We also assessed the impact of viral IL-10 on heme oxygenase 1 (HO-1), which is an enzyme linked with suppression of inflammatory responses. Polarization of monocytes by viral IL-10 resulted in upregulation of HO-1, and inhibition of HO-1 function resulted in a loss of capacity of viral IL-10 to suppress tumor necrosis factor alpha (TNF-α) and IL-1ß, implicating HO-1 in viral IL-10-induced suppression of proinflammatory cytokines by M2c monocytes. In addition, a functional consequence of monocytes polarized with viral IL-10 was a decreased capacity to activate CD4(+) T cells. This study identifies a novel role for viral IL-10 in driving M2c polarization, which may limit virus clearance by restricting proinflammatory and CD4(+) T cell responses at sites of infection.

Differentiated neuroblastoma cells provide a highly efficient model for studies of productive varicella-zoster virus infection of neuronal cells.

Varicella-zoster virus (VZV) is a highly species-specific herpesvirus that targets sensory ganglionic neurons. This species specificity has limited the study of many aspects of VZV pathogenesis, including neuronal infection. We report development of a highly efficient neuroblastoma cell model to study productive VZV infection of neuronal cells. We show that differentiation of SH-SY5Y neuroblastoma cells yields a homogenous population of neuron-like cells that are permissive to the full VZV replicative cycle. These cells supported productive infection by both laboratory and clinical VZV isolates, including the live varicella vaccine. This model may enable rapid identification of genetic determinants facilitating VZV neurotropism.

Experimental models to study varicella-zoster virus infection of neurons.

Varicella zoster virus (VZV) infection results in the establishment of latency in human sensory neurons. Reactivation of VZV leads to herpes zoster which can be followed by persistent neuropathic pain, termed post-herpetic neuralgia (PHN). Humans are the only natural host for VZV, and the strict species specificity of the virus has restricted the development of an animal model of infection which mimics all phases of disease. In order to elucidate the mechanisms which control the establishment of latency and reactivation as well as the effect of VZV replication on neuronal function, in vitro models of neuronal infection have been developed. Currently these models involve culturing and infecting dissociated human fetal neurons, with or without their supporting cells, an intact explant fetal dorsal root ganglia (DRG) model, neuroblastoma cell lines and rodent neuronal cell models. Each of these models has distinct advantages as well as disadvantages, and all have contributed towards our understanding of VZV neuronal infection. However, as yet none have been able to recapitulate the full virus lifecycle from primary infection to latency through to reactivation. The development of such a model will be a crucial step towards advancing our understanding of the mechanisms involved in VZV replication in neuronal cells, and the design of new therapies to combat VZV-related disease.

Snapshot of HIV pathogenesis in China.

Several reviews have focused on the nature of HIV infection and its spread in various geographical regions of China. In contrast, this review provides a comprehensive update on the prevalence of multiple HIV-1 subtypes, consequent emergence of recombinant and novel forms of HIV-1 in China, and the implications this may have on HIV diversity and the development of effective vaccines. In addition it also examines the dissemination of primary drug resistance in therapy naïve patients, as well as co-infections with two other important viruses-hepatitis B and C. The main purpose of this review is to provide a current snapshot of HIV-1 pathogenesis in China and possibly shed some light on the future of HIV evolution, and potential challenges for future vaccine and anti-retroviral therapeutics against HIV strains in this area.

HIV-1 co-infection, superinfection and recombination.

As the human immunodeficiency virus (HIV) pandemic progresses, an increasing number of recombinant viruses have been identified and in many geographical regions they are now the predominating strain. These recombinants are formed when an individual has acquired a co-infection or superinfection with more than one HIV-1 strain or subtype. Thus, dually infected individuals provide opportunities for studying HIV recombinants and viral interactions between infecting strains in vivo. The possible epidemiological, clinical and therapeutic implications of dual infections and recombination are many. Recombination may result in the emergence of more pathogenic and virulent HIV strains with altered fitness, tropism, and resistance to multiple drugs, and may hamper the development of subtype-based vaccines. This review is aimed at providing a more thorough understanding of dual infections (both co-infection and super-infection) and the possible consequences of the emergence of recombinant HIV-1 strains.