Complex patterns of the HIV-1 epidemic in Kuala Lumpur, Malaysia: Evidence for expansion of circulating recombinant form CRF33_01B and detection of multiple other recombinants

AbstractThe HIV protease-reverse transcriptase (PR-RT) (1047 bp), gp120-env (891 bp) and gp41-env (547 bp) regions from the plasma of 115 HIV-1-infected patients in Kuala Lumpur (KL), Malaysia were sequenced. Detailed phylogenetic and bootscanning analyses were performed to determine the mosaic structure of the HIV-1 strains and their recombination breakpoint(s). Among the 50 patient samples in which all three regions could be amplified, the HIV-1 CRF01_AE subtype (46%) was predominant followed by subtypes B (10%) and B′ (6%). A total of 9/50 (18%) patients were infected with a CRF01_AE/B inter-subtype recombinant, displaying a recombinant form (RF)PR-RT, CRF01_AEgp120-env and CRF01_AEgp41-env. This RF was derived from the Thai variants of CRF01_AE and B′ subtype, with two distinct B′ subtype segments in the backbone of CRF01_AE, similar to the newly identified CRF33_01B. In addition, one sample demonstrated a close structural relationship with the new CRF33_01B in the PR-RT region but displayed B′ segment in part of the env region (RFPR-RT, CRF01_AE/B′gp120-env and B′gp41-env) indicating continuing evolution of CRF33_01B. The remaining 18% of samples were identified as unique recombinant forms (URFs)

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.This work was funded by NHMRC project grant APP1088005 awarded to AA, BS and BM. and NHMRC project grant APP1126599 awarded to DT and AA. DT was funded by NHMRC fellowship APP110432

Synthetic long oligonucleotides to generate artificial templates for use as positive controls in molecular assays: drug resistance mutations in influenza virus as an example

<p>Abstract</p> <p>Background</p> <p>Positive controls are an integral component of any sensitive molecular diagnostic tool, but this can be affected, if several mutations are being screened in a scenario of a pandemic or newly emerging disease where it can be difficult to acquire all the necessary positive controls from the host. This work describes the development of a synthetic oligo-cassette for positive controls for accurate and highly sensitive diagnosis of several mutations relevant to influenza virus drug resistance.</p> <p>Results</p> <p>Using influenza antiviral drug resistance mutations as an example by employing the utility of synthetic paired long oligonucleotides containing complementary sequences at their 3' ends and utilizing the formation of oligonucleotide dimers and DNA polymerization, we generated ~170bp dsDNA containing several known specific neuraminidase inhibitor (NAI) resistance mutations. These templates were further cloned and successfully applied as positive controls in downstream assays.</p> <p>Conclusion</p> <p>This approach significantly improved the development of diagnosis of resistance mutations in terms of time, accuracy, efficiency and sensitivity, which are paramount to monitoring the emergence and spread of antiviral drug resistant influenza strains. Thus, this may have a significantly broader application in molecular diagnostics along with its application in rapid molecular testing of all relevant mutations in an event of pandemic.</p

Relating in vitro neutralisation level and protection in the CVnCoV (CUREVAC) trial

Abstract A recent study analysed the relationship between neutralising antibody response and protection from SARS-CoV-2 infection across eight vaccine platforms 1 . The efficacy results from a phase 2b/3 trial of a ninth vaccine candidate, CVnCoV (CUREVAC), was announced on 16 June 2021 2 . The low efficacy of this new mRNA vaccine, which showed only 47% protection from symptomatic SARS-CoV-2 infection, was surprising given the high efficacy of two previous mRNA-based vaccines 3,4 . A number of factors have been suggested to play a role in the low efficacy in the CVnCoV study, particularly around the dose and immunogenicity of the vaccine (which uses an unmodified mRNA construct 5,6 ) and the potential role of infection with SARS-CoV-2 variants (which were the dominant strains observed in the CVnCoV trial) 2

Varicella zoster virus productively infects human natural killer cells and manipulates phenotype.

Varicella zoster virus (VZV) is a ubiquitous human alphaherpesvirus, responsible for varicella upon primary infection and herpes zoster following reactivation from latency. To establish lifelong infection, VZV employs strategies to evade and manipulate the immune system to its advantage in disseminating virus. As innate lymphocytes, natural killer (NK) cells are part of the early immune response to infection, and have been implicated in controlling VZV infection in patients. Understanding of how VZV directly interacts with NK cells, however, has not been investigated in detail. In this study, we provide the first evidence that VZV is capable of infecting human NK cells from peripheral blood in vitro. VZV infection of NK cells is productive, supporting the full kinetic cascade of viral gene expression and producing new infectious virus which was transmitted to epithelial cells in culture. We determined by flow cytometry that NK cell infection with VZV was not only preferential for the mature CD56dim NK cell subset, but also drove acquisition of the terminally-differentiated maturity marker CD57. Interpretation of high dimensional flow cytometry data with tSNE analysis revealed that culture of NK cells with VZV also induced a potent loss of expression of the low-affinity IgG Fc receptor CD16 on the cell surface. Notably, VZV infection of NK cells upregulated surface expression of chemokine receptors associated with trafficking to the skin -a crucial site in VZV disease where highly infectious lesions develop. We demonstrate that VZV actively manipulates the NK cell phenotype through productive infection, and propose a potential role for NK cells in VZV pathogenesis

Characterization of the Host Immune Response in Human Ganglia after Herpes Zoster▿

Varicella-zoster virus (VZV) causes varicella (chicken pox) and establishes latency in ganglia, from where it reactivates to cause herpes zoster (shingles), which is often followed by postherpetic neuralgia (PHN), causing severe neuropathic pain that can last for years after the rash. Despite the major impact of herpes zoster and PHN on quality of life, the nature and kinetics of the virus-immune cell interactions that result in ganglion damage have not been defined. We obtained rare material consisting of seven sensory ganglia from three donors who had suffered from herpes zoster between 1 and 4.5 months before death but who had not died from herpes zoster. We performed immunostaining to investigate the site of VZV infection and to phenotype immune cells in these ganglia. VZV antigen was localized almost exclusively to neurons, and in at least one case it persisted long after resolution of the rash. The large immune infiltrate consisted of noncytolytic CD8+ T cells, with lesser numbers of CD4+ T cells, B cells, NK cells, and macrophages and no dendritic cells. VZV antigen-positive neurons did not express detectable major histocompatibility complex (MHC) class I, nor did CD8+ T cells surround infected neurons, suggesting that mechanisms of immune control may not be dependent on direct contact. This is the first report defining the nature of the immune response in ganglia following herpes zoster and provides evidence for persistence of non-latency-associated viral antigen and inflammation beyond rash resolution

Relating in vitro neutralisation level and protection in the CVnCoV (CUREVAC) trial.

The vaccine candidate CVnCoV (CUREVAC) showed surprisingly low efficacy in a recent phase 3 trial compared with other mRNA vaccines. Here we show that the low efficacy follows from the dose used and the presence of SARS-CoV-2 variants, and is predicted by the neutralising antibody response induced by the vaccine

RHIM-based protein:protein interactions in microbial defence against programmed cell death by necroptosis

The Receptor-interacting protein kinase Homotypic Interaction Motif (RHIM) is an amino acid sequence that mediates multiple protein:protein interactions in the mammalian programmed cell death pathway known as necroptosis. At least one key RHIM-based complex has been shown to have a functional amyloid fibril structure, which provides a stable hetero-oligomeric platform for downstream signaling. RHIMs and related motifs are present in immunity-related proteins across nature, from viruses to fungi to metazoans. Necroptosis is a hallmark feature of cellular clearance of infection. For this reason, numerous pathogens, including viruses and bacteria, have developed varied methods to modulate necroptosis, focusing on inhibiting RHIM:RHIM interactions, and thus their downstream cell death effects. This review will discuss current understanding of RHIM:RHIM interactions in normal cellular activation of necroptosis, from a structural and cell biology perspective. It will compare the mechanisms by which pathogens subvert these interactions in order to maintain their replicative and infective cycles and consider the similarities between RHIMs and other functional amyloid-forming proteins associated with cell death and innate immunity. It will discuss the implications of the heteromeric nature and structure of RHIM-based amyloid complexes in the context of other functional amyloids

The RHIM of the Immune Adaptor Protein TRIF Forms Hybrid Amyloids with Other Necroptosis-Associated Proteins

TIR-domain-containing adapter-inducing interferon-&beta; (TRIF) is an innate immune protein that serves as an adaptor for multiple cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. One outcome of TRIF-directed signalling is the activation of the programmed cell death pathway necroptosis, which is governed by interactions between proteins that contain a RIP Homotypic Interaction Motif (RHIM). TRIF contains a RHIM sequence and can interact with receptor interacting protein kinases 1 (RIPK1) and 3 (RIPK3) to initiate necroptosis. Here, we demonstrate that the RHIM of TRIF is amyloidogenic and supports the formation of homomeric TRIF-containing fibrils. We show that the core tetrad sequence within the RHIM governs the supramolecular organisation of TRIF amyloid assemblies, although the stable amyloid core of TRIF amyloid fibrils comprises a much larger region than the conserved RHIM only. We provide evidence that RHIMs of TRIF, RIPK1 and RIPK3 interact directly to form heteromeric structures and that these TRIF-containing hetero-assemblies display altered and emergent properties that likely underlie necroptosis signalling in response to Toll-like receptor activation

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