COVID-19 causes a clinical spectrum of acute and chronic illness and host / virus interactions are not completely understood1,2. To identify host factors that can influence SARS-CoV-2 infection, we screened the human genome for genes... more
COVID-19 causes a clinical spectrum of acute and chronic illness and host / virus interactions are not completely understood1,2. To identify host factors that can influence SARS-CoV-2 infection, we screened the human genome for genes that, when upregulated, alter the outcome of authentic SARS-CoV-2 infection. From this, we identify 34 new genes that can alter the course of infection, including the innate immune receptor P-selectin, which we show is a novel SARS-CoV-2 spike receptor. At the cellular level expression of P-selectin does not confer tropism for SARS-CoV-2, instead it acts to suppress infection. More broadly, P-selectin can also promote binding to SARS-CoV-2 variants, SARS-CoV-1 and MERS, acting as a general spike receptor for highly pathogenic coronaviruses. P-selectin is expressed on platelets and endothelium3, and we confirm SARS-CoV-2 spike interactions with these cells are P-selectin-dependent and can occur under shear flow conditions.In vivo, authentic SARS-CoV-2 us...
Research Interests: Immunology, Biology, Platelet, Virus, ENDOTHELIUM, and 3 moreTropism, Coronavirus, and Innate Immune System
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Patients with indolent lymphoma undertaking recurrent or continuous B cell suppression are at risk of severe COVID‐19. Patients and healthy controls (HC; N = 13) received two doses of BNT162b2: follicular lymphoma (FL; N = 35) who were... more
Patients with indolent lymphoma undertaking recurrent or continuous B cell suppression are at risk of severe COVID‐19. Patients and healthy controls (HC; N = 13) received two doses of BNT162b2: follicular lymphoma (FL; N = 35) who were treatment naïve (TN; N = 11) or received immunochemotherapy (ICT; N = 23) and Waldenström's macroglobulinemia (WM; N = 37) including TN (N = 9), ICT (N = 14), or treated with Bruton's tyrosine kinase inhibitors (BTKi; N = 12). Anti‐spike immunoglobulin G (IgG) was determined by a high‐sensitivity flow‐cytometric assay, in addition to live‐virus neutralization. Antigen‐specific T cells were identified by coexpression of CD69/CD137 and CD25/CD134 on T cells. A subgroup (N = 29) were assessed for third mRNA vaccine response, including omicron neutralization. One month after second BNT162b2, median anti‐spike IgG mean fluorescence intensity (MFI) in FL ICT patients (9977) was 25‐fold lower than TN (245 898) and HC (228 255, p = .0002 for both). An...
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Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of... more
Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifical...
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BackgroundCancer is associated with excess morbidity and mortality from coronavirus disease 2019 (COVID-19) following infection by the novel pandemic coronavirus SARS-CoV-2. Vaccinations against SARS-CoV-2 have been rapidly developed and... more
BackgroundCancer is associated with excess morbidity and mortality from coronavirus disease 2019 (COVID-19) following infection by the novel pandemic coronavirus SARS-CoV-2. Vaccinations against SARS-CoV-2 have been rapidly developed and proved highly effective in reducing the incidence of severe COVID-19 in clinical trials of healthy populations. However, patients with cancer were excluded from pivotal clinical trials. Early data suggest that vaccine response is less robust in patients with immunosuppressive conditions or treatments, while toxicity and acceptability of COVID-19 vaccines in the cancer population is unknown. Unanswered questions remain about the impact of various cancer characteristics (such as treatment modality and degree of immunosuppression) on serological response to and safety of COVID-19 vaccinations. Furthermore, as the virus and disease manifestations evolve, ongoing data is required to address the impact of new variants.MethodsSerOzNET is a prospective obse...
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RaPID mRNA display was used for the discovery of antiviral cyclic peptides that potently and selectively inhibit SARS-CoV-2 Mpro. The most potent inhibitor exhibited a novel binding mode, interacting with residues across the homodimer... more
RaPID mRNA display was used for the discovery of antiviral cyclic peptides that potently and selectively inhibit SARS-CoV-2 Mpro. The most potent inhibitor exhibited a novel binding mode, interacting with residues across the homodimer interface.
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Understanding the long-term maintenance of SARS-CoV-2 immunity is critical for predicting protection against reinfection. In an age and gender matched cohort of 24 participants, the association of disease severity and early immune... more
Understanding the long-term maintenance of SARS-CoV-2 immunity is critical for predicting protection against reinfection. In an age and gender matched cohort of 24 participants, the association of disease severity and early immune responses on the maintenance of humoral immunity 12 months post-infection is examined. All severely affected participants maintain a stable subset of SARS-CoV-2 receptor-binding domain (RBD)-specific memory B cells (MBCs) and good neutralising antibody breadth against the majority of the variants of concern, including the Delta variant. Modelling these immune responses against vaccine efficacy data indicate 45-76% protection against symptomatic infection (variant dependent). Overall, these findings indicate durable humoral responses in most participants after infection, reasonable protection against reinfection, and implicate baseline antigen-specific CD4+ T cell responses as a predictor of maintenance of antibody neutralisation breadth and RBD-specific MBC levels at 12 months post-infection.
Research Interests: Immunology, Biology, Medicine, Immunity, Immune system, and 2 moreAntibody and Humoral Immunity(Antibody and Humoral Immunity)
(Antibody and Humoral Immunity)
From late 2020 the world observed the rapid emergence of many distinct SARS-CoV-2 variants. At the same time, pandemic responses coalesced into significant global vaccine roll-out that have now significantly lowered Covid-19 hospital and... more
From late 2020 the world observed the rapid emergence of many distinct SARS-CoV-2 variants. At the same time, pandemic responses coalesced into significant global vaccine roll-out that have now significantly lowered Covid-19 hospital and mortality rates in the developed world. Over this period, we developed a rapid platform (R-20) for viral isolation and characterisation using primary remnant diagnostic swabs. This combined with quarantine testing and genomics surveillance, enabled the rapid isolation and characterisation of all major SARS-CoV-2 variants (all variants of concern and 6 variants of interest) globally with a 4-month period. This platform facilitated viral variant isolation and enabled rapid resolution of variant phenotype by allowing determining end point viral titers from primary nasopharyngeal swabs and through ranking of evasion of neutralising antibodies. In late 2021, when the Delta variant was dominating, Omicron rapidly emerged. Using this platform, we isolated ...
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The COVID-19 pandemic caused by the SARS-CoV-2 virus has led to a major public health burden and has resulted in millions of deaths worldwide. As effective treatments are limited, there is a significant requirement for high-throughput,... more
The COVID-19 pandemic caused by the SARS-CoV-2 virus has led to a major public health burden and has resulted in millions of deaths worldwide. As effective treatments are limited, there is a significant requirement for high-throughput, low resource methods for the discovery of novel antivirals. The SARS-CoV-2 spike protein plays a key role in viral entry and has been identified as a therapeutic target. Using the available spike crystal structure, we performed a virtual screen with a library of 527 209 natural compounds against the receptor binding domain of this protein. Top hits from this screen were subjected to a second, more comprehensive molecular docking experiment and filtered for favourable ADMET properties. The in vitro activity of 10 highly ranked compounds was assessed using a virus neutralisation assay designed to facilitate viral entry in a physiologically relevant manner via the plasma membrane route. Subsequently, four compounds ZINC02111387, ZINC02122196, SN00074072 and ZINC04090608 were identified to possess antiviral activity in the µM range. These findings validate the virtual screening method as a tool for identifying novel antivirals and provide a basis for future drug development against SARS-CoV-2.
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SummaryAlthough ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of factors controlling SARS-CoV-2 host interactions has not been described. Here we used whole genome CRISPR activation to identify host... more
SummaryAlthough ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of factors controlling SARS-CoV-2 host interactions has not been described. Here we used whole genome CRISPR activation to identify host factors controlling SARS-CoV-2 Spike binding. The top hit was a Toll-like receptor-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where it forms a cell surface complex with LRRC15 but does not support infection. Instead, LRRC15 functioned as a negative receptor suppressing both pseudotyped and live SARS-CoV-2 infection. LRRC15 is expressed in collagen-producing lung myofibroblasts where it can sequester virus and reduce infection in trans. Mechanistically LRRC15 is regulated by TGF-β, where moderate LRRC15 expression drives collagen production but high levels suppress it, revealing a novel lung fibrosis feedback circuit. Overall, LRRC15 is a mast...
Research Interests: Biology()
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Chronic lymphocytic leukemia (CLL) is associated with immunocompromise and high risk of severe COVID-19 disease and mortality. Monoclonal B-Lymphocytosis (MBL) patients also have immune impairment. We evaluated humoral and cellular immune... more
Chronic lymphocytic leukemia (CLL) is associated with immunocompromise and high risk of severe COVID-19 disease and mortality. Monoclonal B-Lymphocytosis (MBL) patients also have immune impairment. We evaluated humoral and cellular immune responses in 181 patients with CLL (160) and MBL (21) to correlate failed seroconversion (<50AU/mL SARS-CoV-2 II IgG assay, antibody to spike protein, Abbott Diagnostics) following each of 2 vaccine doses with clinical and laboratory parameters. Following first and second doses, 79.2% then 45% of CLL, and 50% then 9.5% of MBL respectively remained seronegative, indicating 2 vaccine doses are crucial. There was significant association between post-dose 2 antibody level with pre-vaccination reduced IgM (p<0.0001), IgG2 (p<0.035), IgG3 (p<0.046), and CLL therapy within 12 months (p<0.001) in univariate analysis. By multivariate analysis, reduced IgM (p<0.0002) and active therapy (p<0.0002) retained significance. There was no signi...
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Serological testing for SARS-CoV-2-specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence and host immune response. A greater understanding of the relationship between... more
Serological testing for SARS-CoV-2-specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. Sera from recovered patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection (n = 200), and negative control sera collected prior to the COVID-19 pandemic (n = 100), were assessed in parallel. Performance was assess...
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The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and... more
The development of a safe and effective vaccine is a key requirement to overcoming the COVID-19 pandemic. Recombinant proteins represent the most reliable and safe vaccine approach but generally require a suitable adjuvant for robust and durable immunity. We used the SARS-CoV-2 genomic sequence and in silico structural modelling to design a recombinant spike protein vaccine (Covax-19™). A synthetic gene encoding the spike extracellular domain (ECD) was inserted into a baculovirus backbone to express the protein in insect cell cultures. The spike ECD was formulated with Advax-SM adjuvant and first tested for immunogenicity in C57BL/6 and BALB/c mice. The Advax-SM adjuvanted vaccine induced high titers of binding antibody against spike protein that were able to neutralise the original wildtype virus on which the vaccine was based as well as the variant B.1.1.7 lineage virus. The Covax-19 vaccine also induced potent spike-specific CD4+ and CD8+ memory T-cells with a dominant Th1 phenotype, and this was shown to be associated with cytotoxic T lymphocyte killing of spike labelled target cells in vivo. Ferrets immunised with Covax-19 vaccine intramuscularly twice 2 weeks apart made spike receptor binding domain (RBD) IgG and were protected against an intranasal challenge with SARS-CoV-2 virus 2 weeks after the second immunisation. Notably, ferrets that received two 25 or 50μg doses of Covax-19 vaccine had no detectable virus in their lungs or in nasal washes at day 3 post-challenge, suggesting the possibility that Covax-19 vaccine may in addition to protection against lung infection also have the potential to block virus transmission. This data supports advancement of Covax-19 vaccine into human clinical trials.
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ABSTRACTThe COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein and affinity ligands to this surface protein... more
ABSTRACTThe COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (KD) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect spike protein in solution by ELISA, and the co-crystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a β-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide liga...
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ABSTRACTAntibodies against coronavirus spike protein potently protect against infection and disease, however it remains unclear if such protection can be extended to variant coronaviruses. This is exemplified by a set of iconic and... more
ABSTRACTAntibodies against coronavirus spike protein potently protect against infection and disease, however it remains unclear if such protection can be extended to variant coronaviruses. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here we explore antibody maturation strategies to change and broaden their specificity, enabling potent binding and neutralization of SARS-CoV-2. Using targeted mutagenesis as well as light chain shuffling on phage, we identified variants with considerably increased affinity and neutralization potential. The most potent antibody, derived from the NIH-developed mAb m396, neutralized live SARS-CoV-2 virus with a half-maximal inhibitory concentration (IC50) of 160 ng/ml. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specifici...
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ABSTRACTAs the COVID-19 pandemic evolves, human milk banks worldwide continue to provide donor human milk to vulnerable infants who lack access to mother’s own milk. Under these circumstances, ensuring the safety of donor human milk is... more
ABSTRACTAs the COVID-19 pandemic evolves, human milk banks worldwide continue to provide donor human milk to vulnerable infants who lack access to mother’s own milk. Under these circumstances, ensuring the safety of donor human milk is paramount, as the risk of vertical transmission of SARS-CoV-2 is not well understood. Here, we investigate the inactivation of SARS-CoV-2 in human milk by pasteurisation, and the stability of SARS-CoV-2 in human milk under cold storage (freezing or refrigeration). Following heating to 63°C or 56°C for 30 minutes, SARS-CoV-2 replication competent (i.e. live) virus was undetected in both human milk and the control medium. Cold storage of SARS-CoV-2 in human milk (either at 4°C or - 30°C) did not significantly impact infectious viral load over a 48 hour period. Our findings demonstrate that SARS-CoV-2 can be effectively inactivated by Holder pasteurisation, and confirm that existing milk bank processes will effectively mitigate the risk of transmission o...
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Uncoating of the metastable HIV-1 capsid is a tightly regulated disassembly process required for release of the viral cDNA prior to nuclear import. To understand the intrinsic capsid disassembly pathway and how it can be modulated, we... more
Uncoating of the metastable HIV-1 capsid is a tightly regulated disassembly process required for release of the viral cDNA prior to nuclear import. To understand the intrinsic capsid disassembly pathway and how it can be modulated, we have developed a single-particle fluorescence microscopy method to follow the real-time uncoating kinetics of authentic HIV capsids immediately after permeabilizing the viral membrane. Opening of the first defect in the lattice is the rate-limiting step of uncoating, which is followed by rapid, catastrophic collapse. The capsid-binding inhibitor PF74 accelerates capsid opening but stabilizes the remaining lattice. In contrast, binding of a polyanion to a conserved arginine cluster in the lattice strongly delays initiation of uncoating but does not prevent subsequent lattice disassembly. Our observations suggest that different stages of uncoating can be controlled independently with the interplay between different capsid-binding regulators likely to det...
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T follicular helper (Tfh) cells are increasingly recognized as a major reservoir of HIV infection that will likely need to be addressed in approaches to curing HIV. However, Tfh express minimal CCR5, the major coreceptor for HIV-1, and... more
T follicular helper (Tfh) cells are increasingly recognized as a major reservoir of HIV infection that will likely need to be addressed in approaches to curing HIV. However, Tfh express minimal CCR5, the major coreceptor for HIV-1, and the mechanism by which they are infected is unclear. We have previously shown that macaque Tfh lack CCR5, but are infected in vivo with CCR5-using SIV at levels comparable to other memory CD4(+) T cells. Similarly, human splenic Tfh cells are highly infected with HIV-1 DNA. Therefore, we set out to examine the mechanism of infection of Tfh cells. Tfh and other CD4(+) T cell subsets from macaque lymph nodes and spleens, splenic Tfh from HIV(+) subjects, and tonsillar Tfh from HIV-uninfected subjects were isolated by cell sorting prior to cell surface and molecular characterization. HIV proviral gp120 sequences were submitted to genotypic and phenotypic tropism assays. Entry of CCR5- and CXCR4-using viruses into Tfh from uninfected tonsillar tissue was ...
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Transcriptional gene silencing (TGS) of mammalian genes can be induced by short interfering RNA (siRNA) targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA), which targets tandem NF-κB motifs within the... more
Transcriptional gene silencing (TGS) of mammalian genes can be induced by short interfering RNA (siRNA) targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA), which targets tandem NF-κB motifs within the viral 5'LTR. In this study, we screened a siRNA panel with the aim of identifying novel 5'LTR targets, to provide multiplexing potential with enhanced viral silencing and application toward developing alternate therapeutic strategies. Systematic examination identified a novel siRNA target, si143, confirmed to induce TGS as the silencing mechanism. TGS was prolonged with virus suppression >12 days, despite a limited ability to induce post- TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by chromatin immunoprecipitation analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac, and recruitment of argonaute-1, all characteristic marks of ...
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Epidermal Langerhans cells (eLCs) uniquely express the C-type lectin receptor langerin in addition to the HIV entry receptors CD4 and CCR5. They are among the first target cells to encounter HIV in the anogenital stratified squamous... more
Epidermal Langerhans cells (eLCs) uniquely express the C-type lectin receptor langerin in addition to the HIV entry receptors CD4 and CCR5. They are among the first target cells to encounter HIV in the anogenital stratified squamous mucosa during sexual transmission. Previous reports on the mechanism of HIV transfer to T cells and the role of langerin have been contradictory. In this study, we examined HIV replication and langerin-mediated viral transfer by authentic immature eLCs and model Mutz-3 LCs. eLCs were productively infected with HIV, whereas Mutz-3 LCs were not susceptible because of a lack of CCR5 expression. Two successive phases of HIV viral transfer to T cells via cave/vesicular trafficking and de novo replication were observed with eLCs as previously described in monocyte-derived or blood dendritic cells, but only first phase transfer was observed with Mutz-3 LCs. Langerin was expressed as trimers after cross-linking on the cell surface of Mutz-3 LCs and in this form ...
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ABSTRACT
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Dendritic cells play a major role in HIV pathogenesis. Epithelial dendritic cells appear to be one of the first cells infected after sexual transmission and transfer of the virus to CD4 lymphocytes, simultaneously activating these cells... more
Dendritic cells play a major role in HIV pathogenesis. Epithelial dendritic cells appear to be one of the first cells infected after sexual transmission and transfer of the virus to CD4 lymphocytes, simultaneously activating these cells to produce high levels of HIV replication. Such transfer may occur locally in inflamed mucosa or after dendritic cells have matured and migrated to local lymph nodes. Therefore, the mechanism of binding, internalization, infection and transfer of HIV to CD4 lymphocytes is of great interest. Recently, the role of the C-type lectin DC-SIGN as a dendritic cell receptor for HIV has been intensively studied with in vitro monocyte-derived dendritic cells. However, it is clear that other C-type lectin receptors such as Langerin on Langerhan cells and mannose receptor on dermal dendritic cells are at least equally important for gp120 binding on epithelial dendritic cells. C-type lectin receptors play a role in virus transfer to T cells, either via de novo in...
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Langerhans cells (LCs) are the resident dendritic cells (DCs) of epidermis in human mucosal stratified squamous epithelium and the skin. A phenotypically similar DC has recently been discovered as a minor population in the murine dermis.... more
Langerhans cells (LCs) are the resident dendritic cells (DCs) of epidermis in human mucosal stratified squamous epithelium and the skin. A phenotypically similar DC has recently been discovered as a minor population in the murine dermis. In epidermis, LCs function as sentinel antigen‐presenting cells that can capture invading viruses such as herpes simplex virus (HSV), varicella‐zoster virus (VZV) and human immunodeficiency virus (HIV). This interaction between LCs and viruses results in highly variable responses, depending on the virus as discussed in this review. For example, HSV induces apoptosis in LCs but HIV does not. LCs seem to be the first in a complex chain of antigen presentation to T cells in lymph nodes for HSV and possibly VZV, or they transport virus to T cells, as described for HIV and maybe VZV. Together with epidermal keratinocytes they may also have a role in the initial innate immune response at the site of infection in the epidermis, although this is not fully k...