Induction of human immunodeficiency virus type 1-specific T cells by a bluetongue virus tubule-vectored vaccine prime-recombinant modified virus Ankara boost regimen.

In the absence of strategies for reliable induction of antibodies broadly neutralizing human immunodeficiency virus type 1 (HIV-1), vaccine efforts have shifted toward the induction of cell-mediated immunity. Here we describe the construction and immunogenicity of novel T-cell vaccine NS1.HIVA, which delivers the HIV-1 clade A consensus-derived immunogen HIVA on the surface of tubular structures spontaneously formed by protein NS1 of bluetongue virus. We demonstrated that NS1 tubules can accommodate a protein as large as 527 amino acids without losing their self-assembly capability. When injected into BALB/c mice by several routes, chimeric NS1.HIVA tubules induced HIV-1-specific major histocompatibility complex class I-restricted T cells. These could be boosted by modified virus Ankara expressing the same immunogen and generate a memory capable of gamma interferon (IFN-gamma) production, proliferation, and lysis of sensitized target cells. Induced memory T cells readily produced IFN-gamma 230 days postimmunization, and upon a surrogate virus challenge, NS1.HIVA vaccine alone decreased the vaccinia virus vv.HIVA load in ovaries by 2 orders of magnitude 280 days after immunization. Thus, because of its T-cell immunogenicity and antigenic simplicity, the NS1 delivery system could serve as a priming agent for heterologous prime-boost vaccination regimens. Its usefulness in primates, including humans, remains to be determined

rAAV.sFlt-1 Gene Therapy Achieves Lasting Reversal of Retinal Neovascularization in the Absence of a Strong Immune Response to the Viral Vector

PURPOSE. To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy. METHODS. trVEGF029 mice were injected subretinally with rAAV.sFlt-1 or phosphate-buffered saline. Fluorescein angiography and electroretinography were used to compare the extent of fluorescein leakage from retinal vessels and retinal function, respectively. A group of eyes was enucleated, and the retinal vasculature and morphology were studied by confocal and light microscopy. Cells were isolated from the posterior eyecups and spleens of a further group, and immune cell subset populations were investigated by flow cytometry. sFlt-1 protein levels in the eyes were evaluated by ELISA. RESULTS. After a single rAAV.sFlt-1 injection, sFlt-1 protein levels were upregulated, and there was a reduction in fluorescein leakage from the retinal vessels and an improvement in retinal function. Confocal microscopy of isolectin-IB4-labeled retinal wholemounts showed more normal-appearing capillary beds in rAAV.sFlt-1-injected than in PBS-injected trVEGF029 mouse eyes. Light microscopy demonstrated retinal morphology preservation, with fewer aberrant vessels invading the outer nuclear layer of rAAV.sFlt-1-injected eyes. Furthermore, the immune response to subretinal injection of rAAV.sFlt-1 was limited to a transient increase in CD45 ϩ leukocytes that disappeared by 4 weeks after injection. This transient increase was localized to the eye and did not affect long-term therapeutic efficacy. CONCLUSIONS. The data support the notion that rAAV.sFlt-1 gene therapy is safe and effective for the long-term inhibition of deleterious blood vessel growth in the eye. (Invest Ophthalmol Vis Sci. 2009;50:4279 -4287

OPTIMUM study protocol: an adaptive randomised controlled trial of a mixed whole-cell/acellular pertussis vaccine schedule.

INTRODUCTION: Combination vaccines containing whole-cell pertussis antigens were phased out from the Australian national immunisation programme between 1997 and 1999 and replaced by the less reactogenic acellular pertussis (aP) antigens. In a large case-control study of Australian children born during the transition period, those with allergist diagnosed IgE-mediated food allergy were less likely to have received whole-cell vaccine in early infancy than matched population controls (OR: 0.77 (95% CI, 0.62 to 0.95)). We hypothesise that a single dose of whole-cell vaccine in early infancy is protective against IgE-mediated food allergy. METHODS AND ANALYSIS: This adaptive double-blind randomised controlled trial is investigating whether a mixed whole-cell/aP vaccine schedule prevents allergic disease in the first year of life. The primary outcome is IgE-mediated food allergy by 12 months of age. Secondary outcomes include new onset of atopic dermatitis by 6 or 12 months of age; sensitisation to at least one allergen by 12 months of age; seroconversion in anti-pertussis toxin IgG titres after vaccination with aP booster at 18 months of age; and solicited systemic and local adverse events following immunisation with pertussis-containing vaccines. Analyses will be performed using a Bayesian group sequential design. ETHICS AND DISSEMINATION: This study has been approved by the Child and Adolescent Health Service Human Research Ethics Committee, Perth, Western Australia (RGS 00019). The investigators will ensure that this trial is conducted in accordance with the principles of the Declaration of Helsinki and with the International Conference on Harmonisation Guidelines for Good Clinical Practice. Individual consent will be requested. Parents will be reimbursed reasonable travel and parking costs to attend the study visits. The dissemination of these research findings will follow the National Health and Medical Research Council of Australia Open Access Policy. TRIAL REGISTRATION NUMBER: ACTRN12617000065392p

Innate immunity defines the capacity of antiviral T cells to limit persistent infection

Effective immunity requires the coordinated activation of innate and adaptive immune responses. Natural killer (NK) cells are central innate immune effectors, but can also affect the generation of acquired immune responses to viruses and malignancies. How NK cells influence the efficacy of adaptive immunity, however, is poorly understood. Here, we show that NK cells negatively regulate the duration and effectiveness of virus-specific CD4+ and CD8+ T cell responses by limiting exposure of T cells to infected antigen-presenting cells. This impacts the quality of T cell responses and the ability to limit viral persistence. Our studies provide unexpected insights into novel interplays between innate and adaptive immune effectors, and define the critical requirements for efficient control of viral persistence

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

OPTIMUM study protocol: an adaptive randomised controlled trial of a mixed whole-cell/acellular pertussis vaccine schedule

Introduction Combination vaccines containing whole-cell pertussis antigens were phased out from the Australian national immunisation programme between 1997 and 1999 and replaced by the less reactogenic acellular pertussis (aP) antigens. In a large case–control study of Australian children born during the transition period, those with allergist diagnosed IgE-mediated food allergy were less likely to have received whole-cell vaccine in early infancy than matched population controls (OR: 0.77 (95% CI, 0.62 to 0.95)). We hypothesise that a single dose of whole-cell vaccine in early infancy is protective against IgE-mediated food allergy.Methods and analysis This adaptive double-blind randomised controlled trial is investigating whether a mixed whole-cell/aP vaccine schedule prevents allergic disease in the first year of life. The primary outcome is IgE-mediated food allergy by 12 months of age. Secondary outcomes include new onset of atopic dermatitis by 6 or 12 months of age; sensitisation to at least one allergen by 12 months of age; seroconversion in anti-pertussis toxin IgG titres after vaccination with aP booster at 18 months of age; and solicited systemic and local adverse events following immunisation with pertussis-containing vaccines. Analyses will be performed using a Bayesian group sequential design.Ethics and dissemination This study has been approved by the Child and Adolescent Health Service Human Research Ethics Committee, Perth, Western Australia (RGS 00019). The investigators will ensure that this trial is conducted in accordance with the principles of the Declaration of Helsinki and with the International Conference on Harmonisation Guidelines for Good Clinical Practice. Individual consent will be requested. Parents will be reimbursed reasonable travel and parking costs to attend the study visits. The dissemination of these research findings will follow the National Health and Medical Research Council of Australia Open Access Policy.Trial registration number ACTRN12617000065392p

Prime–boost immunization generates a high frequency, high-avidity CD8+ cytotoxic T lymphocyte population

Development and expansion of high-avidity T cell populations may be important for the success of immunization strategies against HIV and other pathogens that have presented major problems for vaccine development. We have used tetrameric–MHC complexes ex vivo and lytic assays to show that `prime-boost' immunization with DNA vaccines and recombinant poxvirus vectors generates high frequencies of cytotoxic T lymphocytes (CTL) that recognize target cells expressing very low levels of specific antigen. These cells persist for at least 6 months at levels representing ~10% of the CD8+ T cell population. Using a novel in vivo assay, we also found that prime-boost immunized animals were capable of eliminating target cells expressing 10- to 100-fold less immunogenic peptide than mice given either vector alone. In addition, viral challenge led to rapid expansion of CTL effectors in prime-boost groups, to levels representing >30% of total CD8+ T cell numbers. Strategies that generate specific T cells of high avidity, optimizing early detection of infected cells, offer new hope for effective prophylaxis and immunotherapy

CpG pretreatment enhances antiviral T-cell immunity against cytomegalovirus.

Major histocompatibility complex class I-restricted T-cell immunity is essential to control infection with cytomegalovirus (CMV), a clinically important virus that causes significant disease in immunocompromised individuals. Cross-presentation is considered the primary mode of antigen presentation to generate protective antiviral CD8(+) T-cell immunity. Herpesviruses, including CMV, encode numerous proteins that interfere with direct antigen presentation, leading to the paradigm that T-cell immunity to these pathogens necessitates cross-presentation. However, the antigen presentation requirements needed to generate a protective T-cell response to CMV remain unknown. Here, we show that a fully functional antiviral CD8(+) T-cell response can be generated in a system where cross-presentation is shut down by pretreatment with CpG. Notably, in this setting, CD8(+) T cells demonstrate accelerated control of infection, and organ pathology is limited. These data indicate that protective antiviral T-cell immunity to CMV is generated by direct presentation and can be enhanced by pretreatment with CpG

rAAV.sFlt-1 gene therapy achieves lasting reversal of retinal neovascularization in the absence of a strong immune response to the viral vector

PURPOSE. To determine the efficacy of rAAV.sFlt-1-mediated gene therapy in a transgenic mouse model of retinal neovascularization (trVEGF029) and to assess whether rAAV.sFlt-1 administration generated any deleterious, long-lasting immune response that could affect efficacy