Dimeric IgA is a specific biomarker of recent SARS-CoV-2 infection.

Current tests for SARS-CoV-2 antibodies (IgG, IgM, IgA) cannot differentiate recent and past infections. We describe a point of care, lateral flow assay for SARS-CoV-2 dIgA based on the highly selective binding of dIgA to a chimeric form of secretory component (CSC), that distinguishes dIgA from monomeric IgA. Detection of specific dIgA uses a complex of biotinylated SARS-CoV-2 receptor binding domain and streptavidin-colloidal gold. SARS-CoV-2-specific dIgA was measured both in 112 cross-sectional samples and a longitudinal panel of 362 plasma samples from 45 patients with PCR-confirmed SARS-CoV-2 infection, and 193 discrete pre-COVID-19 or PCR-negative patient samples. The assay demonstrated 100% sensitivity from 11 days post-symptom onset, and a specificity of 98.2%. With an estimated half-life of 6.3 days, dIgA provides a unique biomarker for the detection of recent SARS-CoV-2 infections with potential to enhance diagnosis and management of COVID-19 at point-of-care.

Frequent occurrence of low-level positive autoantibodies in chronic hepatitis C.

Evidence of autoimmune disease associated with hepatitis C virus (HCV)-infection has important clinical implications. A systematic profile of these autoantibodies in relevant clinical cohorts relative to healthy controls is needed to better inform current standard of care for chronic hepatitis C. Samples from an Australian cohort of chronic HCV-infected subjects (n=127) were tested for the presence of 19 diagnostic autoantibodies and compared with data available from a control cohort representing a general Caucasian population (n=198). Chronic HCV-infected individuals had a greater number of autoantibodies than controls (p<0.0001). Anti-nuclear antibodies (ANA) followed by anti-smooth muscle antibodies (SMA) were the most frequently detected autoantibodies within the HCV cohort and significantly more than in the control cohort (p<0.0001 and p=0.006, respectively). However, for most autoantibody assays the 95th percentile approximated the reference value for positivity. None of the autoantibodies were significantly associated with age or sex for the HCV cohort, except SMA positivity that was significantly higher in chronic HCV-infected male subjects (p<0.0001). Autoantibodies found in chronic HCV-infected subjects were commonly low positive and not disease-specific. Accordingly, general screening for autoimmunity in HCV-infected subjects should not be performed unless there is high clinical suspicion of an underlying autoimmune disease.

Successful elevation of circulating acetate and propionate by dietary modulation does not alter T-regulatory cell or cytokine profiles in healthy humans: a pilot study.

PURPOSE: Increased circulating concentrations of short-chain fatty acids (SCFA) achieved by ingestion of high-fibre diets is associated with anti-inflammatory effects through promotion of FoxP3+ regulatory T(reg) cells in mouse models. This study aimed to determine whether similar increments in blood SCFA levels can be achieved in humans and whether these are associated with similar immune modulatory effects. METHODS: In a pilot single-blinded, randomised, controlled cross-over study in ten healthy subjects, the effects were determined of high- (39 g/day) and low-fibre (18 g/day) intake (all food provided) on SCFA (gas chromatography), proportions of Treg cells (flow cytometry) and a panel of cytokines (multiplex methodology) measured in peripheral blood at day 5 of each diet. RESULTS: Actual fibre intake differed between the diets by 19 [16-21] g/day (P< 0.001). Median [range] total plasma SCFA levels with high-fibre intake were 174.5 [104.8-249.5] µmol/L, which were greater than those associated with low-fibre intake at 59.0 [26.5-79.9] (P < 0.001). Differences were significantly different for both acetate and propionate. The frequencies of total CD4 T cells and T-regulatory cells, and concentrations of inflammatory and anti-inflammatory cytokines were not significantly different between the dietary interventions. CONCLUSIONS: Plasma SCFA levels can be modulated by altering dietary fibre consumption in healthy individuals with increments similar to those achieved in murine studies. Five days of diet intervention did not result in changes in regulatory T-cell proportions and cytokine concentrations in peripheral blood, and may require longer duration of dietary change.

Contemporary analysis of functional immune recovery to opportunistic and vaccine-preventable infections after allogeneic haemopoietic stem cell transplantation.

OBJECTIVES: Infections are a major cause of mortality after allogeneic haemopoietic stem cell transplantation (alloHSCT), and immune recovery is necessary for prevention. Novel transplant procedures have changed the epidemiology of infections but contemporary data on functional immune recovery are limited. In this pilot study, we aimed to measure immune recovery in the current era of alloHSCT. METHODS: Twenty, 13, 11, 9 and 9 alloHSCT recipients had blood collected at baseline (time of conditioning) and 3-, 6-, 9-, and 12-months post-alloHSCT, respectively. Clinical data were collected, and immune recovery was measured using immunophenotyping, lymphocyte proliferation, cytokine analysis and antibody isotyping. RESULTS: Median absolute T- and B-cell counts were below normal from baseline until 9- to 12-months post-alloHSCT. Median absolute CD4+ T-cell counts recovered at 12-months post-alloHSCT. Positive proliferative responses to Aspergillus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), influenza and tetanus antigens were detected from 9 months. IL-6 was the most abundant cytokine in cell cultures. In cultures stimulated with CMV, EBV, influenza and tetanus peptides, the CD4+ T-cell count correlated with IL-1ß (P = 0.045) and CD8+ T-cell count with IFNγ (P = 0.013) and IL-1ß (P = 0.012). The NK-cell count correlated with IL-1ß (P = 0.02) and IL-17a (P = 0.03). Median serum levels of IgG1, IgG2 and IgG3 were normal while IgG4 and IgA were below normal range throughout follow-up. CONCLUSIONS: This pilot study demonstrates that immune recovery can be measured using CD4+ T-cell counts, in vitro antigen stimulation and selected cytokines (IFNγ, IL-1ß, IL-4, IL-6, IL-17, IL-21, IL-31) in alloHSCT recipients. While larger studies are required, monitoring immune recovery may have utility in predicting infection risk post-alloHSCT.

Detection of HCV-Specific IFN-γ Responses in HCV Antibody and HCV RNA Negative Injecting Drug Users.

BACKGROUND: Detectable HCV-specific cellular immune responses in HCV antibody and RNA negative people who inject drugs (PWID) raise the question of whether some are resistant to HCV infection. Immune responses from people who have been exposed to hepatitis C virus (HCV) and remain anti-HCV negative are of interest for HCV vaccine development; however, limited research addresses this area. OBJECTIVES: In a cohort of HCV antibody and RNA negative PWID, we assessed whether the presence of HCV-specific IFN-γ responses or genetic associations provide any evidence of protection from HCV infection. PATIENTS AND METHODS: One hundred and ninety-eight participants were examined longitudinally for clinical, behavioral, social, environmental and genetic characteristics (IFNL3 genotype [formally IL-28B] and HLA type). Sixty-one of the 198 participants were HCV antibody and RNA negative, with 53 able to be examined longitudinally for HCV-specific IFN-γ ELISpot T cell responses. RESULTS: Ten of the 53 HCV antibody and RNA negative participants had detectable HCV-specific IFN-γ responses at baseline (18%). The magnitude of IFN-γ responses averaged 131 +/- 96 SFC/106 PBMC and the breadth was mean 1 +/- 1 pool positive. The specificity of responses were mainly directed to E2, NS4b and NS5b. Participants with (10) and without (43) HCV-specific IFN-γ responses did not differ in behavioral, clinical or genetic characteristics (P > 0.05). There was a larger proportion sharing needles (with 70%, without 49%, P = 0.320) and a higher incidence of HCV (with 35.1 per 100 py, 95% CI 14.6, 84.4, without 16.0 per 100 py, 95% CI 7.2, 35.6, P = 0.212) in those with IFN-γ responses, although not statistically significant. Half the participants with baseline IFN-γ responses became HCV RNA positive (5/10), with one of these participants spontaneously clearing HCV. The spontaneous clearer had high magnitude and broad Th1 responses, favorable IFNL3 genotype and favorable HLA types. CONCLUSIONS: This study demonstrated the detection of HCV-specific IFN-γ responses in HCV antibody and RNA negative individuals, with a tendency for HCV-specific IFN-γ responses to be associated with HCV exposure. The potential role of HCV-specific IFN-γ responses in those who remained HCV RNA negative is of value for the development of novel HCV therapeutics.

Maintenance of Th1 hepatitis C virus (HCV)-specific responses in individuals with acute HCV who achieve sustained virological clearance after treatment.

BACKGROUND AND AIM: T-cell responses against hepatitis C are believed to be critical in achieving both natural and treatment-induced clearance. However, rapid clearance of antigen with early treatment of primary infection may result in reduced or poorly sustained cellular immunity. This study longitudinally examined Th1 and Th2 hepatitis C virus (HCV)-specific cytokine production and T-cell effector function from subjects enrolled in the Australian Trial in Acute Hepatitis C comparing three groups: treatment-induced clearance (sustained virological response [SVR]), treatment non-response, and untreated spontaneous clearance. METHODS: HCV-specific T-cell responses were characterized by HCV peptide ELISpot, in vitro cytokine production, and T-cell flow cytometry assays. RESULTS: Treated subjects with a sustained virological response (SVR) displayed a better maintenance of HCV-specific Th1 responses compared to treatment non-responders (higher interferon [IFN]-γ and interleukin (IL)-2 magnitude at week 24, broader IFN-γ responses at weeks 24 and 48, P < 0.05) and significantly increased IFN-γ responses between screening and week 48 (magnitude P = 0.026, breadth P = 0.009). Treatment-induced viral clearance was also associated with a trend toward decreased IL-10 responses (screening to week 48, P = 0.070), higher expression of CD45RO (P = 0.042) and CD38 (P = 0.088) on CD4+ T cells, and higher IFN-γR expression (CD56+ IFN-γR+ P = 0.033) compared to treatment non-responders. Untreated subjects with viral clearance also displayed high magnitude and broad HCV-specific IFN-γ and IL-2 responses early in infection; however, IFN-γ responses were not as well maintained compared to treated subjects with a SVR (week 48 magnitude, breadth P = 0.064). CONCLUSION: Treatment-induced viral clearance of recent HCV infection is associated with maintenance of HCV-specific Th1 responses.

Impaired hepatitis C virus (HCV)-specific interferon-γ responses in individuals with HIV who acquire HCV infection: correlation with CD4(+) T-cell counts.

Studies examining the effect of coinfection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) on the HCV-specific immune response in acute HCV infection are limited. This study directly compared acute HCV-specific T-cell responses and cytokine profiles between 20 HIV/HCV-coinfected and 20 HCV-monoinfected subjects, enrolled in the Australian Trial in Acute Hepatitis C (ATAHC), using HCV peptide enzyme-linked immunospot (ELISPOT) and multiplex in vitro cytokine production assays. HIV/HCV coinfection had a detrimental effect on the HCV-specific cytokine production in acute HCV infection, particularly on HCV-specific interferon γ (IFN-γ) production (magnitude P = .004; breadth P = .046), which correlated with peripheral CD4(+) T-cell counts (ρ = 0.605; P = .005) but not with detectable HIV viremia (ρ = 0.152; P = .534).

Incidence of primary hepatitis C infection and risk factors for transmission in an Australian prisoner cohort.

BACKGROUND: Hepatitis C virus (HCV) infection is common in prisoner populations, particularly those with a history of injecting drug use (IDU). Previous studies of HCV incidence have been based on small case numbers and have not distinguished risk events in prison from those in the community. METHODS: HCV incidence was examined in a longitudinal cohort of 488 Australian prisoners with a history of IDU and documented to be seronegative within 12 months prior to enrollment. Inmates were tested for anti-HCV antibodies and viremia, and interviewed about demographic and behavioral risk factors for transmission. RESULTS: The cohort was predominantly male (65%) with high rates of prior imprisonment (72%) and tattooing (73%), as well as longstanding IDU (mean 8.5 years). Ninety-four incident HCV cases were identified (incidence 31.6 per 100 person years). Independent associations were observed between incident infection and prior imprisonment (p = 0.02) and tattooing (p = 0.03), and surprisingly also with methadone maintenance treatment (MMT) (p < 0.001). CONCLUSIONS: High rates of new HCV infection were found in this prisoner cohort reflecting their substantive risk behavior profile, despite having remained uninfected for many years. The association with MMT is challenging and highlights the need for better understanding of prison-specific HCV transmission risks, as well as the uptake and effectiveness of prevention programs.

A phase I clinical trial of dendritic cell immunotherapy in HCV-infected individuals.

BACKGROUND & AIMS: HCV patients who fail conventional interferon-based therapy have limited treatment options. Dendritic cells are central to the priming and development of antigen-specific CD4(+) and CD8(+) T cell immunity, necessary to elicit effective viral clearance. The aim of the study was to investigate the safety and efficacy of vaccination with autologous dendritic cells loaded with HCV-specific cytotoxic T cell epitopes. METHODS: We examined the potential of autologous monocyte-derived dendritic cells (MoDC), presenting HCV-specific HLA A2.1-restricted cytotoxic T cell epitopes, to influence the course of infection in six patients who failed conventional therapy. Dendritic cells were loaded and activated ex vivo with lipopeptides. In this phase 1 dose escalation study, all patients received a standard dose of cells by the intradermal route while sequential patients received an increased dose by the intravenous route. RESULTS: No patient showed a severe adverse reaction although all experienced transient minor side effects. HCV-specific CD8(+) T cell responses were enumerated in PBMC by ELIspot for interferon-gamma. Patients generated de novo responses, not only to peptides presented by the cellular vaccine but also to additional viral epitopes not represented in the lipopeptides, suggestive of epitope spreading. Despite this, no increases in ALT levels were observed. However, the responses were not sustained and failed to influence the viral load, the anti-HCV core antibody response and the level of circulating cytokines. CONCLUSIONS: Immunotherapy using autologous MoDC pulsed with lipopeptides was safe, but was unable to generate sustained responses or alter the outcome of the infection. Alternative dosing regimens or vaccination routes may need to be considered to achieve therapeutic benefit.

The effects of ALV003 pre-digestion of gluten on immune response and symptoms in celiac disease in vivo.

Effective treatment of celiac disease is an unmet medical need. A glutenase that destroys immunogenic gluten peptides may be clinically valuable. Twenty patients with celiac disease were randomly assigned to ingest a large gluten meal (16 g daily for 3 days) pre-treated with ALV003, a mixture of highly specific glutenases (n=10), or pre-treated with placebo (n=10). Peripheral blood T-cell IFN-gamma ELISpot responses to gliadin and an immunogenic 33mer and symptoms were assessed. While baseline IFN-gamma ELISpot responses to gliadin and the 33mer were negative in all patients, a significant ELISpot response to gliadin or the 33mer was observed in 6 of 10 patients consuming placebo-treated gluten and 0 of 10 consuming ALV003 pre-treated gluten (p=0.011). Symptoms typically associated with gluten ingestion occurred in both groups and were not significantly reduced by ALV003 pre-treatment. ALV003 pre-treatment can abolish immune responses induced by gluten in patients with celiac disease.

Heterosubtypic T-cell responses against avian influenza H5 haemagglutinin are frequently detected in individuals vaccinated against or previously infected with human subtypes of influenza.

BACKGROUND: Cellula r immune responses play a critical role in providing help for the production of neutralizing antibodies to influenza virus, as well as producing anti-viral cytokines and killing infected cells in the lung. Heterosubtypic T-cell responses between different subtypes of influenza have been shown to exist in humans and to provide protection against morbidity and mortality associated with H5N1 infection in animal challenge models. Therefore, existing T-cell responses induced by natural infection or vaccination in humans may provide some degree of protection from infection with H5N1 strains, or may attenuate the severity of disease. OBJECTIVES: To investigate heterosubtypic T-cell responses to avian influenza in humans. METHODS: T-cell responses to an overlapping set of H5 HA peptides and inactivated viruses (H1N1, H3N2 and H5N1) were assessed using IFN-gamma and IL-2 enzyme-linked immunospot (ELISpot) assays in a cohort of adults either vaccinated against seasonal influenza in the last 3 years (n = 20) or previously infected (n = 40). RESULTS: T-cell responses to all three subtypes of virus were found in both infected and vaccinated individuals by IFN-gamma and IL-2 ELISpot assays. Approximately half of the participants from each group had a positive T-cell response to the H5 HA peptides in the IFN-gamma or IL-2 ELISpot assay. CONCLUSIONS: Heterosubtypic T-cell responses to H5 HA occur quite frequently in vaccinated and infected individuals. Further investigation of these responses and what role they may play upon challenge or vaccination against H5N1 may assist in vaccine design for avian influenza.

Host and viral factors in the immunopathogenesis of primary hepatitis C virus infection.

Individuals infected with hepatitis C virus (HCV) have two possible outcomes of infection, clearance or persistent infection. The focus of this review is the host mechanisms that facilitate clearance. The interaction between HCV viral components and the immune system ultimately determines the balance between the virus and host. Strong evidence points to the aspects of cellular immune response as the key determinants of outcome. The recent discovery of viral evasion strategies targeting innate immunity suggests that the interferon-alpha/beta induction pathways are also critical. A growing body of evidence has implicated polymorphisms in both innate and adaptive immune response genes as determinants of viral clearance in individuals infected with HCV.

A randomized, placebo-controlled phase I trial of DNA prime, recombinant fowlpox virus boost prophylactic vaccine for HIV-1.

An HIV-vaccine consisting of a DNA prime, recombinant fowlpox virus (rFPV) boost was evaluated in a double-blind placebo controlled trial. One milligram of pHIS-HIV-B expressing mutated gag, pol, env, vpu, tat and rev was administered at weeks 0 and 4 boosted by 5 x 10(7) pfu rFPV-HIV-B expressing gag/pol at week 8. The vaccine regimen was safe, but there was no difference between vaccine (n = 18) and placebo recipients (n = 6) for Gag or Pol-specific T-cell immune responses at week 9.

Protective immunity against hepatitis C virus infection.

There is increasing evidence that a small percentage of individuals exposed to the hepatitis C virus have the capacity to generate a strong cellular immune response against the virus and avoid persistent infection, and perhaps do so repeatedly after re-exposure. This article reviews the evidence that the responses identified in this unique group of individuals represent the protective immunity that will need to be elicited by hepatitis C virus vaccines.

Prolonged illness after infectious mononucleosis is associated with altered immunity but not with increased viral load.

BACKGROUND: Primary Epstein-Barr virus (EBV) infection causes a spectrum of characteristics that range from asymptomatic seroconversion to severe infectious mononucleosis (IM), sometimes with prolonged symptoms and disability. We examined the relationships between clinical course, number of viral copies, and immunological parameters in a prospective cohort of subjects with recent IM. METHODS: Eight case patients with at least 6 months of disabling symptoms and 31 matched control subjects who had recovered promptly were included. Symptom scores were recorded at regular intervals over the course of 12 months. Cellular EBV load, EBV-specific antibody responses, lymphocyte subsets, and EBV-specific interferon (IFN)- gamma induction were measured. RESULTS: In case patients with prolonged illness, the severity of acute-phase symptoms was greater, the development of anti-EBV nuclear antigen-1 immunoglobulin G was more rapid, and the time to development of the peak IFN- gamma response to the majority of latent-cycle EBV peptides was generally slower than those in control subjects. However, in both groups, neither viral nor immune parameters correlated with the severity or duration of symptoms. CONCLUSIONS: The resolution of symptomatic IM is not determined by control of viremia, nor is it easily explained by altered host responses to EBV infection. The detailed determinants of delayed recovery remain to be elucidated.

Development of a synthetic consensus sequence scrambled antigen HIV-1 vaccine designed for global use.

Induction of high levels of broadly reactive cytotoxic T lymphocytes (CTL) remains a promising approach for an effective HIV-1 vaccine. We have developed a novel genetic-based vaccine strategy that encodes consensus overlapping peptide sets from all HIV-1 proteins scrambled together. This synthetic scrambled antigen vaccine (SAVINE) strategy has significant advantages, e.g. capacity to encode more antigens safely and is very flexible compared to traditional isolate-based strategies. The SAVINE vaccine strategy is clearly immunogenic, being able to restimulate a range of human HIV-1 specific responses in vitro and induce HIV-1 specific immunity in vivo in mice. Interestingly, different in vivo delivery strategies affected the resulting immunity and immunodominance pattern in mice. This platform strategy could be used for other infections and cancers where T cell responses are important for protection.

Evaluation in macaques of HIV-1 DNA vaccines containing primate CpG motifs and fowlpoxvirus vaccines co-expressing IFNgamma or IL-12.

Induction of HIV-specific T-cell responses by vaccines may facilitate efficient control of HIV. Plasmid DNA vaccines and recombinant fowlpoxvirus (rFPV) vaccines are promising HIV-1 vaccine candidates, although either vaccine alone may be insufficient to protect against HIV-1. A consecutive immunisation strategy involving priming with DNA and boosting with rFPV vaccines encoding multiple common HIV-1 antigens was further evaluated in 30 macaques. The DNA vaccine vector included CpG immunostimulatory molecules, and rFPV vaccines were compared with rFPV vaccines co-expressing the pro-T cell cytokines IFNgamma or IL-12. Vaccines expressed multiple HIV-1 genes, mutated to remove active sites of the HIV proteins. The vaccines were well tolerated, and a significant enhancement of DNA-vaccine primed HIV-1 specific T lymphocyte responses was observed following rFPV boosting. Co-expression of IFNgamma or IL-12 by the rFPV vaccines did not further enhance immune responses. Non-sterilising protection from a non-pathogenic HIV-1 challenge was observed. This study provides evidence of a safe, optimised, strategy for the generation of T-cell mediated immunity to HIV-1.

Prevalence of production of virus-specific interferon-gamma among seronegative hepatitis C-resistant subjects reporting injection drug use.

This report describes subjects who were highly likely to have been repeatedly exposed to hepatitis C virus (HCV) through injection drug use and who remained negative for anti-HCV antibody. Production of virus-specific interferon- gamma by peripheral blood mononuclear cells was seen in the majority of subjects (72%) and was associated with higher-risk behavior. For 92% of the subjects, results of recombinant immunoblot assays demonstrated faint bands against nonstructural proteins. The immune responses described are likely to have been primed and maintained by episodes of subclinical infection without classic seroconversion and may indicate a hepatitis C-resistant phenotype. Vaccine strategies to mimic this response may provide protection against persistent HCV infection.

Clearance of hepatitis C viremia associated with cellular immunity in the absence of seroconversion in the hepatitis C incidence and transmission in prisons study cohort.

Understanding the earliest virological and immunological events in acute hepatitis C virus (HCV) infection may provide insight into the determinants of protective immunity. Four cases of HCV viremia with subsequent viral clearance, but without biochemical hepatitis or anti-HCV seroconversion, are reported from a prospective cohort study of prison inmates. Two of the subjects who developed sustained viremia were assessed for production of interferon (IFN)- gamma, by use of the enzyme-linked immunospot (ELISPOT) method and by assessment of HCV cytotoxic T lymphocyte (CTL) activity, CD4 lymphocyte proliferative responses, HCV load, and genotype. After 2-6 months of viremia, all 4 subjects cleared serum HCV RNA. Specific cellular responses were detected in both of the subjects who were assessed, and production of IFN- gamma was demonstrated in one subject. All subjects had weak, but consistent, serological reactivity against HCV nonstructural proteins on immunoblot testing, despite repeatedly nonreactive HCV ELISA tests. These cases highlight the potential for cellular immune responses against HCV to facilitate viral clearance, responses that may model those required for effective HCV vaccination.