Perforin Expression Directly Ex Vivo by HIV-Specific CD8+ T-Cells Is a Correlate of HIV Elite Control

Many immune correlates of CD8+ T-cell-mediated control of HIV replication, including polyfunctionality, proliferative ability, and inhibitory receptor expression, have been discovered. However, no functional correlates using ex vivo cells have been identified with the known ability to cause the direct elimination of HIV-infected cells. We have recently discovered the ability of human CD8+ T-cells to rapidly upregulate perforin—an essential molecule for cell-mediated cytotoxicity—following antigen-specific stimulation. Here, we examined perforin expression capability in a large cross-sectional cohort of chronically HIV-infected individuals with varying levels of viral load: elite controllers (n = 35), viremic controllers (n = 29), chronic progressors (n = 27), and viremic nonprogressors (n = 6). Using polychromatic flow cytometry and standard intracellular cytokine staining assays, we measured perforin upregulation, cytokine production, and degranulation following stimulation with overlapping peptide pools encompassing all proteins of HIV. We observed that HIV-specific CD8+ T-cells from elite controllers consistently display an enhanced ability to express perforin directly ex vivo compared to all other groups. This ability is not restricted to protective HLA-B haplotypes, does not require proliferation or the addition of exogenous factors, is not restored by HAART, and primarily originates from effector CD8+ T-cells with otherwise limited functional capability. Notably, we found an inverse relationship between HIV-specific perforin expression and viral load. Thus, the capability of HIV-specific CD8+ T-cells to rapidly express perforin defines a novel correlate of control in HIV infection

HIV-Specific T-Cells Accumulate in the Liver in HCV/HIV Co-Infection

BACKGROUND AND AIMS: Hepatitis C Virus (HCV)-related liver disease progresses more rapidly in individuals co-infected with Human Immunodeficiency Virus-1 (HIV), although the underlying immunologic mechanisms are unknown. We examined whether HIV-specific T-cells are identified in the liver of HCV/HIV co-infected individuals and promote liver inflammation through bystander immune responses. METHODS: Ex-vivo intra-hepatic lymphocytes from HCV mono-infected and HCV/HIV co-infected individuals were assessed for immune responses to HIV and HCV antigens by polychromatic flow cytometry. RESULTS: HCV/HIV liver biopsies had similar frequencies of lymphocytes but lower percentages of CD4+ T-cells compared to HCV biopsies. In co-infection, intra-hepatic HIV-specific CD8+ and CD4+ T-cells producing IFN-gamma and TNF-alpha were detected and were comparable in frequency to those that were HCV-specific. In co-infected individuals, viral-specific CD8+ T-cells produced more of the fibrogenic cytokine, TNF-alpha. In both mono- and co-infected individuals, intra-hepatic HCV-specific T-cells were poorly functional compared to HIV-specific T-cells. In co-infection, HAART was not associated with a reconstitution of intra-hepatic CD4+ T-cells and was associated with reduction in both HIV and HCV-specific intra-hepatic cytokine responses. CONCLUSION: The accumulation of functional HIV-specific T-cells in the liver during HCV/HIV co-infection may represent a bystander role for HIV in inducing faster progression of liver disease

Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1–infected individuals to a mean of 49.4 ± SD 12.9% of CD8+ T cells expressing Tim-3 in HIV-1–infected chronic progressors versus 28.5 ± 6.8% in HIV-1–uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1–infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4+ T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1–specific CD8+ T cells. Tim-3–expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1–specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1–associated T cell dysfunction

Evaluating Clinical and Immunologic Correlates of HIV Shedding at Mucosal Sites

HIV infects over 33 million people worldwide with a new infection occurring every 9 seconds. Sex is the primary mode of transmission and the majority of new infections occur during unprotected sexual contact between an HIV-infected individual and an uninfected sexual partner(s) since HIV infected individuals tend to shed virus in their genital secretions. The infectiousness of an individual is closely tied to the amount of virus in blood, which is closely associated with HIV levels shed in semen or vaginal fluid or rectal secretions. Although, Highly Active Antiretroviral Therapy (HAART) is associated with complete suppression of HIV RNA in blood to undetectable levels, the impact of HAART on semen HIV RNA levels is less clear. I evaluated the correlation between systemic and mucosal HIV-specific CD8+ T cell immune responses and HIV RNA levels in blood and semen. Overall, there was a strong positive correlation between HIV RNA levels in blood and semen. Neither systemic nor mucosal (in semen) HIV-specific CD8+ responses were associated with HIV RNA levels in blood or semen, in fact CD8+ T cell immune responses in semen correlated with increased HIV RNA levels in semen. Furthermore, inflammatory cytokines (IL-6, and IL-8) CMV levels in semen were associated with increased semen HIV RNA shedding. HAART initiation was associated with complete suppression of HIV viremia, but a significant proportion of individuals on suppressive HAART continue to shed HIV RNA in semen even after 6 months, and this isolated virus was infectious and often present at high levels (> 5000 copies/mL). Nevertheless, long-term HAART was associated with complete immune reconstitution of CD4+ T cells in the sigmoid colon of HIV-infected individuals on long-term therapy. These findings demonstrate that neither systemic nor mucosal HIV-specific CD8+ responses, when assayed with IFN- production as an endpoint, were associated with reduced HIV RNA levels in blood or semen. Semen HIV RNA levels did correlate with local inflammatory cytokines and CMV reactivation. Furthermore, despite effective HAART a significant proportion of HIV-infected men continued to shed HIV RNA in semen. However, long-term completely suppressive HAART was associated with complete immune reconstitution of the sigmoid colon.Ph

Impact of collection method on assessment of semen HIV RNA viral load.

The blood HIV RNA viral load is the best-defined predictor of HIV transmission, in part due to ease of measurement and the correlation of blood and genital tract (semen or cervico-vaginal) viral load, although recent studies found semen HIV RNA concentration to be a stronger predictor of HIV transmission. There is currently no standardized method for semen collection when measuring HIV RNA concentration. Therefore, we compared two collection techniques in order to study of the impact of antiretroviral therapy on the semen viral load.Semen was collected by masturbation from HIV-infected, therapy-naïve men who have sex with men (MSM) either undiluted (Visit 1) or directly into transport medium (Visit 2). Seminal plasma was then isolated, and the HIV RNA concentration obtained with each collection technique was measured and corrected for dilution if necessary. Collection of semen directly into transport medium resulted in a median HIV RNA viral load that was 0.4 log10 higher than undiluted samples.The method of semen collection is an important consideration when quantifying the HIV RNA viral load in this compartment

Quantitative Analysis of SARS-CoV-2 Serological Responses Post Three Doses of Immunization and Prior to Breakthrough COVID-19 Infections

Background: Vaccine mediated SARS-CoV-2 antibody responses should be carefully evaluated. With regular follow-up in healthy individuals, we aimed to determine SARS-CoV-2 serological responses post three doses of immunization and prior to breakthrough infections in the Canadian population. Methods: In a prospective cohort study, we enrolled 140 healthy participants post COVID-19 vaccination in Kingston, Ontario, Canada. IgG antibodies against the SARS-CoV-2 spike receptor–binding domain were quantified by immunoassay post three doses of immunization. With COVID-19 rapid antigen test, polymerase chain reaction, and whole genome sequencing, 27 breakthrough infections were identified. Results: Following SARS-CoV-2 vaccine (including BNT162b2, AZD1222, and mRNA-1273), the median serum anti-spike protein antibody level was 143.6 BAU/mL (binding antibody unit, interquartile range 79.0–266.6) post the first dose of immunization, 1046.4 BAU/mL (423.9–1738.2) post the second dose, and 1604.7 BAU/mL (700.1–3764.0) post the third dose. Observed differences were significant (p ≤ 0.001). The median antibody level of 1604.7 BAU/mL post third dose is 45.6 times that of the seroconversion level (35.2 BAU/mL). This indicates that most vaccines approved are effective in producing robust antibody responses. In seven breakthrough cases characterized by whole genome sequencing, prior to infection, antibody concentrations of breakthrough cases were at 3249.4 (Delta), 2748.4 (Delta), 4893.9 (Omicron), 209.1 (Omicron), and 231.5 (Omicron), 725.7 (Omicron), and 2346.6 (Omicron) BAU/mL. Compared with the average antibody concentration of 2057.7 BAU/mL (58 times that of the seroconversion concentration) from above seven cases, 37.2% of triple vaccinated, 19.0% of double vaccinated, and 1.5% single dosed individuals have higher SARS-CoV-2 antibody levels. Conclusions: Most vaccines are effective in producing robust antibody responses when more than one dose is given, and the more doses the higher the serological response. Likely due to the highly contagious nature of SARS-CoV-2 variants, a significant number of participants have SARS-CoV-2 antibody responses lower than the average antibody concentration prior to the known breakthrough infections. Additional vaccination is likely required to ensure immunity against infection by SARS-CoV-2

Molecular characterization of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae in Ontario, Canada, 2008-2011.

Due to the lack of detailed reports of Klebsiella pneumoniae carbapenemase (KPC)-producing enterobacteria in Ontario, Canada, we perform a molecular characterization of KPC-producing Enterobacteriaceae submitted to the provincial reference laboratory from 2008 to 2011. Susceptibility profiles were accessed by E-test. Molecular types of isolates were determined by pulse-field gel electrophoresis (PFGE) and multilocus sequence typing. Screening of ß-lactamase genes was performed by multiplex PCR and alleles were identified by DNA sequencing. The genetic platform of blaKPC gene was analyzed by PCR. Plasmid replicons were typed using PCR-based typing approach. KPC-plasmids were also evaluated by S1 nuclease-PFGE and Southern blot. Thirty unique clinical isolates (26 Klebsiella pneumoniae, 2 Enterobacter cloacae, 1 Citrobacter freundii and 1 Raoultella ornithinolytica) were identified as blaKPC positive: 4 in 2008, 3 in 2009, 10 in 2010 and 13 in 2011. The majority exhibited resistance to carbapenems, cephalosporins and fluoroquinolones and two isolates were also resistant to colistin. The isolates harbored blaKPC-2 (n = 23) or blaKPC-3 (n = 7). blaTEM-1 (n = 27) was commonly detected and occasionally blaOXA-1 (n = 3) and blaCTX-M-15 (n = 1). As expected, all K. pneumoniae isolates carried blaSHV-11. blaKPC genes were identified on Tn4401a (n = 20) or b (n = 10) isoforms, on plasmids of different sizes belonging to the incompatibility groups IncFIIA (n = 19), IncN (n = 3), IncI2 (n = 3), IncFrep (n = 2) and IncA/C (n = 1). The occurrence of KPC ß-lactamase in Ontario was mainly associated with the spread of the K. pneumoniae clone ST258

Isolation, Identification, and Characterization of a Serine Protease with Anti-nociceptive Activity from the Gut Bacterium, Faecalibacterium prausnitzii

Inflammatory bowel disease (IBD) induces hyperexcitability in dorsal root ganglion (DRG) nociceptors, which contributes to abdominal pain. Proteases modulate the excitability of nociceptive neurons via activation of cell-surface protease-activated receptors. Recent findings indicate that serine proteases from Faecalibacterium prausnitzii, a bacterial strain found in the colonic microbiota of healthy individuals, can directly signal to the DRG neurons and reduce excitability, which may suppress abdominal pain. Herein we identify an anti-nociceptive serine protease from F. prausnitzii by heterologous expression and purification of multiple putative anti-nociceptive serine proteases followed by clamp electrophysiology experiments. A single serine protease, Clp-fp, from F. prausnitzii reduced DRG neuronal excitability via activation of protease-activated receptor-4, thereby recapitulating the effect of F. prausnitzii culture supernatants. The identification and characterization of a potentially analgesic serine protease from a commensal gut bacterium makes possible the further exploration of new treatments for IBD-related abdominal pain

Blood and semen HIV RNA viral load.

<p>Blood was collected and the HIV RNA viral load assayed the same way at both study visits (BVL1 and BVL2, respectively); semen was collected undiluted at visit 1 (SVL1) and directly into transport medium and visit 2 (SVL2). Participants with an undetectable semen viral load at both study visits were excluded from statistical analysis.</p

A Bioluminescent Biosensor for Quantifying the Interaction of SARS-CoV-2 and Its Receptor ACE2 in Cells and In Vitro

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is currently spreading and mutating with increasing speed worldwide. Therefore, there is an urgent need for a simple, sensitive, and high-throughput (HTP) assay to quantify virus–host interactions in order to quickly evaluate the infectious ability of mutant viruses and to develop or validate virus-inhibiting drugs. Here, we developed an ultrasensitive bioluminescent biosensor to evaluate virus–cell interactions by quantifying the interaction between the SARS-CoV-2 receptor binding domain (RBD) and its cellular receptor angiotensin-converting enzyme 2 (ACE2) both in living cells and in vitro. We have successfully used this novel biosensor to analyze SARS-CoV-2 RBD mutants and evaluated candidate small molecules (SMs), antibodies, and peptides that may block RBD:ACE2 interaction. This simple, rapid, and HTP biosensor tool will significantly expedite the detection of viral mutants and the anti-COVID-19 drug discovery process.Medicine, Faculty ofNon UBCMedicine, Department ofNeurology, Division ofReviewedFacult