Monkeypox in Montréal: Epidemiology, Phylogenomics, and Public Health Response to a Large North American Outbreak.

BACKGROUND: Monkeypox, a viral zoonotic disease, is causing a global outbreak outside of endemic areas. OBJECTIVE: To characterize the outbreak of monkeypox in Montréal, the first large outbreak in North America. DESIGN: Epidemiologic and laboratory surveillance data and a phylogenomic analysis were used to describe and place the outbreak in a global context. SETTING: Montréal, Canada. PATIENTS: Probable or confirmed cases of monkeypox. MEASUREMENTS: Epidemiologic, clinical, and demographic data were aggregated. Whole-genome sequencing and phylogenetic analysis were performed for a set of outbreak sequences. The public health response and its evolution are described. RESULTS: Up to 18 October 2022, a total of 402 cases of monkeypox were reported mostly among men who have sex with men (MSM), most of which were suspected to be acquired through sexual contact. All monkeypox genomes nested within the B.1 lineage. Montréal Public Health worked closely with the affected communities to control the outbreak, becoming the first jurisdiction to offer 1 dose of the Modified Vaccinia Ankara-Bavarian Nordic vaccine as preexposure prophylaxis (PrEP) to those at risk in early June 2022. Two peaks of cases were seen in early June and July (43 and 44 cases per week, respectively) followed by a decline toward near resolution of the outbreak in October. Reasons for the biphasic peak are not fully elucidated but may represent the tempo of vaccination and/or several factors related to transmission dynamics and case ascertainment. LIMITATIONS: Clinical data are self-reported. Limited divergence among sequences limited genomic epidemiologic conclusions. CONCLUSION: A large outbreak of monkeypox occurred in Montréal, primarily among MSM. Successful control of the outbreak rested on early and sustained engagement with the affected communities and rapid offer of PrEP vaccination to at-risk persons. PRIMARY FUNDING SOURCE: None.

Persistence of infectivity in elderly individuals diagnosed with severe acute respiratory coronavirus virus 2 (SARS-CoV-2) infection 10 days after onset of symptoms: A cross-sectional study.

We performed viral culture of nasopharyngeal specimens in individuals aged 79 and older, infected with severe acute respiratory coronavirus virus 2 (SARS-CoV-2), 10 days after symptom onset. A positive viral culture was obtained in 10 (45%) of 22 participants, including 4 (33%) of 12 individuals with improving symptoms. The results of this small study suggest that infectivity may be prolonged among older individuals.

Progressive disseminated histoplasmosis in an apparently immunocompetent patient with a bioprosthetic aortic valve.

Histoplasma capsulatum is an endemic fungus in eastern Canada. This organism has a wide spectrum of manifestations ranging from isolated pulmonary to disseminated disease. The latter usually occurs in immunocompromised hosts or those with substantial environmental exposure. In rare instances, disseminated histoplasmosis can present as an endovascular infection or chronic progressive disseminated histoplasmosis. If not recognized, these entities are almost uniformly fatal. We report a case of an immunocompetent man with a history of longstanding constitutional symptoms. An infectious cause was initially presumed to be unlikely given the chronic nature of his presentation and an extensive series of negative investigations. A diagnosis was only obtained post-mortem upon the unusual detection of both yeast and hyphal forms in blood culture bottles inoculated with a bone marrow aspirate.

L'Histoplasma capsulatum est une mycose endémique dans l'est du Canada. Cet organisme présente un vaste spectre de manifestations, de l'atteinte pulmonaire isolée à la maladie disséminée. En général, la forme disséminée s'observe chez des hôtes immunodéprimés ou qui y ont été largement exposés dans l'environnement. Dans de rares cas, l'histoplasmose disséminée peut prendre la forme d'une infection endovasculaire ou d'une histoplasmose disséminée progressive chronique. Non diagnostiquées, ces entités sont pratiquement toujours fatales. Les auteurs déclarent le cas d'un homme ayant une histoire de symptômes constitutionnels de longue date. Au départ, la cause infectieuse était considérée comme peu probable étant donné la présentation chronique et la longue série d'explorations négatives. Le diagnostic n'a été posé qu'après le décès, lors de la détection inhabituelle des formes à levures et à mycélium dans les flacons d'hémoculture inoculés d'un aspirat de moelle osseuse.

DNA Vaccine-Encoded Flagellin Can Be Used as an Adjuvant Scaffold to Augment HIV-1 gp41 Membrane Proximal External Region Immunogenicity.

Flagellin's potential as a vaccine adjuvant has been increasingly explored over the last three decades. Monomeric flagellin proteins are the only known agonists of Toll-like receptor 5 (TLR5). This interaction evokes a pro-inflammatory state that impacts upon both innate and adaptive immunity. While pathogen associated molecular patterns (PAMPs) like flagellin have been used as stand-alone adjuvants that are co-delivered with antigen, some investigators have demonstrated a distinct advantage to incorporating antigen epitopes within the structure of flagellin itself. This approach has been particularly effective in enhancing humoral immune responses. We sought to use flagellin as both scaffold and adjuvant for HIV gp41 with the aim of eliciting antibodies to the membrane proximal external region (MPER). Accordingly, we devised a straightforward step-wise approach to select flagellin-antigen fusion proteins for gene-based vaccine development. Using plasmid DNA vector-based expression in mammalian cells, we demonstrate robust expression of codon-optimized full length and hypervariable region-deleted constructs of Salmonella enterica subsp. enterica serovar Typhi flagellin (FliC). An HIV gp41 derived sequence including the MPER (gp41607-683) was incorporated into various positions of these constructs and the expressed fusion proteins were screened for effective secretion, TLR5 agonist activity and adequate MPER antigenicity. We show that incorporation of gp41607-683 into a FliC-based scaffold significantly augments gp41607-683 immunogenicity in a TLR5 dependent manner and elicits modest MPER-specific humoral responses in a mouse model.

Development of a DNA vaccine expressing a secreted HIV-1 gp41 ectodomain that includes the membrane-proximal external region.

A limited number of sites on the HIV-1 Envelope protein are vulnerable to broadly neutralizing antibodies (bnAbs). One of these sites, the membrane proximal external region (MPER), is located at the C-terminus of the gp41 ectodomain (gp41ecto). This highly conserved sequence is bound by several well-characterized bnAbs. Efforts to produce a gp41 immunogen are in part hampered by the MPER's hydrophobicity and propensity to induce aggregation. We sought to produce a DNA vaccine expressing a gp41ecto that is both secreted from mammalian cells and maintains binding by bnAbs to the MPER. Through in silico analysis, we predicted regions of gp41ecto that could induce aggregation and possibly hinder secretion. We generated deletion mutants of gp41ecto and tested their ability to be secreted by mammalian cells. Upon deletion of regions in either the fusion peptide (FP) or MPER, secretion of the gp41ecto increased. In an effort to both augment secretion and maintain binding by bnAbs, we developed constructs with the FP deletion and introduced point mutations in the MPER. Two constructs (gp41 ΔFP and gp41 ΔFP+I682E) maintained binding by gp41 MPER-specific bnAbs (4E10, Z13e1 and 10E8). These were evaluated as DNA vaccines in a mouse model. Both vaccines proved to be immunogenic and appeared to elicit some MPER-specific antibodies that bound gp41 ectodomain-derived proteins but not short peptides spanning the MPER. No neutralizing capacity was detected against a clade C virus containing a homologous MPER.

Update on rilpivirine: a new potent non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV replication.

INTRODUCTION: A combination of antiretroviral drugs (ARVs) is necessary to achieve sustained virologic suppression of HIV viral load (< 50 copies/mL). Rilpivirine (RPV) is a potent new non-nucleoside reverse transcriptase inhibitor (NNRTI) that has the potential to be part of effective ARV combinations. Here, we review currently available data on RPV from the standpoint of virologic suppression and efficacy, drug-drug interactions safety, and resistance. AREAS COVERED: This review presents data on the results of clinical trials involving RPV. The topics considered include antiviral potency, dosing, clinical utility, drug resistance, toxicity profile, and pharmacokinetics. EXPERT OPINION: RPV is a potent new addition to the antiretroviral family of drugs for use in combination therapy in previously untreated HIV-infected patients. However, caution needs to be exercised in administration of RPV to patients who initiated therapy with viral loads > 100,000 viral RNA copies/mL.

HIV-1 and influenza antigens synthetically linked to IgG2a Fc elicit superior humoral responses compared to unmodified antigens in mice.

Using murine IgG subclass molecules (IgG1 or IgG2a) synthetically fused to HIV-1 or influenza test antigens, we explored the potential for IgG Fc scaffolds to augment immunogenicity. Each antigen (Ag) was grafted onto a hinge-Fc scaffold containing all critical residues necessary for interaction with effector cells, thus retaining effector functions of the native IgG subclass. We hypothesized that the differential affinity of FcγRs for specific IgG subclasses would influence the magnitude of immune responses elicited by immunization with an Ag-IgG Fc fusion vaccine. We demonstrate here that the antigen-specific humoral response elicited by Ag-IgG2a fusion vaccines is at least tenfold greater than that elicited by native antigen, that this response is superior to that elicited by Ag-IgG1, and that the augmented antigen-specific humoral response elicited is Fcγ receptor-dependent.

Phase 1 safety and immunogenicity evaluation of ADMVA, a multigenic, modified vaccinia Ankara-HIV-1 B'/C candidate vaccine.

BACKGROUND: We conducted a Phase I dose-escalation trial of ADMVA, a Clade-B'/C-based HIV-1 candidate vaccine expressing env, gag, pol, nef, and tat in a modified vaccinia Ankara viral vector. Sequences were derived from a prevalent circulating HIV-1 recombinant form in Yunnan, China, an area of high HIV incidence. The objective was to evaluate the safety and immunogenicity of ADMVA in human volunteers. METHODOLOGY/PRINCIPAL FINDINGS: ADMVA or placebo was administered intramuscularly at months 0, 1 and 6 to 50 healthy adult volunteers not at high risk for HIV-1. In each dosage group [1x10(7) (low), 5x10(7) (mid), or 2.5x10(8) pfu (high)] volunteers were randomized in a 3:1 ratio to receive ADMVA or placebo in a double-blinded design. Subjects were followed for local and systemic reactogenicity, adverse events including cardiac adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA, immunoflourescent staining, and HIV-1 neutralization. Cellular immunogenicity was assessed by a validated IFNgamma ELISpot assay and intracellular cytokine staining. Anti-vaccinia binding titers were measured by ELISA. ADMVA was generally well-tolerated, with no vaccine-related serious adverse events or cardiac adverse events. Local or systemic reactogenicity events were reported by 77% and 78% of volunteers, respectively. The majority of events were of mild intensity. The IFNgamma ELISpot response rate to any HIV antigen was 0/12 (0%) in the placebo group, 3/12 (25%) in the low dosage group, 6/12 (50%) in the mid dosage group, and 8/13 (62%) in the high dosage group. Responses were often multigenic and occasionally persisted up to one year post vaccination. Antibodies to gp120 were detected in 0/12 (0%), 8/13 (62%), 6/12 (50%) and 10/13 (77%) in the placebo, low, mid, and high dosage groups, respectively. Antibodies persisted up to 12 months after vaccination, with a trend toward agreement with the ability to neutralize HIV-1 SF162 in vitro. Two volunteers mounted antibodies that were able to neutralize clade-matched viruses. CONCLUSIONS/SIGNIFICANCE: ADMVA was well-tolerated and elicited durable humoral and cellular immune responses. TRIAL REGISTRATION: Clinicaltrials.gov NCT00252148.

alpha-defensins released into stimulated CD8+ T-cell supernatants are likely derived from residual granulocytes within the irradiated allogeneic peripheral blood mononuclear cells used as feeders.

We recently demonstrated the ability of human beta-defensins to inhibit HIV-1 replication in vitro and demonstrated that alpha-defensins account for the great majority of beta-chemokine independent antiretroviral activity in stimulated CD8+ T-cell culture supernatants. In a follow-up study aimed at defining specific subpopulations of CD8+ T-cells that produce alpha-defensins, we have found that in the absence of irradiated allogeneic peripheral blood mononuclear cells (PBMC), stimulated CD8+ T-cell supernatants do not contain alpha-defensins. In our present work, we define residual granulocytes within PBMC fractions as the likely source. In addition, we describe in vitro conditions that promote the internalization of alpha-defensins by cells not natively producing these proteins, thus confounding our ability to define true alpha-defensin producer cells. In light of these findings, alpha-defensins released into stimulated CD8+ T-cell supernatants are unlikely to be derived from the CD8+ T-cells themselves. Moreover, our data imply that under some experimental conditions, a soluble noncytolytic anti-HIV-1 factor other than beta-chemokines is either not produced by CD8+ T-cells or is present in too small quantity to be effective.