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Objective To systematically review associations between intake of saturated fat and trans unsaturated fat and all cause mortality, cardiovascular disease (CVD) and associated mortality, coronary heart disease (CHD) and associated mortality, ischemic stroke, and type 2 diabetes.Design Systematic review and meta-analysis.Data sources Medline, Embase, Cochrane Central Registry of Controlled Trials, Evidence-Based Medicine Reviews, and CINAHL from inception to 1 May 2015, supplemented by bibliographies of retrieved articles and previous reviews.Eligibility criteria for selecting studies Observational studies reporting associations of saturated fat and/or trans unsaturated fat (total, industrially manufactured, or from ruminant animals) with all cause mortality, CHD/CVD mortality, total CHD, ischemic stroke, or type 2 diabetes.Data extraction and synthesis Two reviewers independently extracted data and assessed study risks of bias. Multivariable relative risks were pooled. Heterogeneity was assessed and quantified. Potential publication bias was assessed and subgroup analyses were undertaken. The GRADE approach was used to evaluate quality of evidence and certainty of conclusions.Results For saturated fat, three to 12 prospective cohort studies for each association were pooled (five to 17 comparisons with 90 501-339 090 participants). Saturated fat intake was not associated with all cause mortality (relative risk 0.99, 95% confidence interval 0.91 to 1.09), CVD mortality (0.97, 0.84 to 1.12), total CHD (1.06, 0.95 to 1.17), ischemic stroke (1.02, 0.90 to 1.15), or type 2 diabetes (0.95, 0.88 to 1.03). There was no convincing lack of association between saturated fat and CHD mortality (1.15, 0.97 to 1.36; P=0.10). For trans fats, one to six prospective cohort studies for each association were pooled (two to seven comparisons with 12 942-230 135 participants). Total trans fat intake was associated with all cause mortality (1.34, 1.16 to 1.56), CHD mortality (1.28, 1.09 to 1.50), and total CHD (1.21, 1.10 to 1.33) but not ischemic stroke (1.07, 0.88 to 1.28) or type 2 diabetes (1.10, 0.95 to 1.27). Industrial, but not ruminant, trans fats were associated with CHD mortality (1.18 (1.04 to 1.33) v 1.01 (0.71 to 1.43)) and CHD (1.42 (1.05 to 1.92) v 0.93 (0.73 to 1.18)). Ruminant trans-palmitoleic acid was inversely associated with type 2 diabetes (0.58, 0.46 to 0.74). The certainty of associations between saturated fat and all outcomes was “very low.” The certainty of associations of trans fat with CHD outcomes was “moderate” and “very low” to “low” for other associations.Conclusions Saturated fats are not associated with all cause mortality, CVD, CHD, ischemic stroke, or type 2 diabetes, but the evidence is heterogeneous with methodological limitations. Trans fats are associated with all cause mortality, total CHD, and CHD mortality, probably because of higher levels of intake of industrial trans fats than ruminant trans fats. Dietary guidelines must carefully consider the health effects of recommendations for alternative macronutrients to replace trans fats and saturated fats.

SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC 50 ) values as low as 9 × 10 − 14 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2. Here, the authors combine three different antibody specificities and an Fc domain on a single multivalent molecule, resulting in high neutralization activity despite viral sequence variability.

Since its emergence in Wuhan, China, in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected ≈6 million persons worldwide. As SARS-CoV-2 spreads across the planet, we explored the range of human cells that can be infected by this virus. We isolated SARS-CoV-2 from 2 infected patients in Toronto, Canada; determined the genomic sequences; and identified single-nucleotide changes in representative populations of our virus stocks. We also tested a wide range of human immune cells for productive infection with SARS-CoV-2. We confirm that human primary peripheral blood mononuclear cells are not permissive for SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor single-nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine viral genotype and phenotype by using in vitro and in vivo infection models.

Provision of pasteurized donor human milk, as a bridge to mother’s own milk, is the standard of care for very low-birth-weight infants in hospital. The aim of this research was to confirm that Holder pasteurization (62.5°C for 30 min) would be sufficient to inactivate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in donated human milk samples. We spiked frozen milk samples from 10 donors to the Rogers Hixon Ontario Human Milk Bank with SARS-CoV-2 to achieve a final concentration of 1 × 107 TCID50/mL (50% of the tissue culture infectivity dose per mL). We pasteurized samples using the Holder method or held them at room temperature for 30 minutes and plated serial dilutions on Vero E6 cells for 5 days. We included comparative controls in the study using milk samples from the same donors without addition of virus (pasteurized and unpasteurized) as well as replicates of Vero E6 cells directly inoculated with SARS-CoV-2. We reported cytopathic effects as TCID50/mL. We detected no cytopathic activity in any of the SARS-CoV-2–spiked milk samples that had been pasteurized using the Holder method. In the SARS-CoV-2–spiked milk samples that were not pasteurized but were kept at room temperature for 30 minutes, we observed a reduction in infectious viral titre of about 1 log. Pasteurization of human milk by the Holder method (62.5°C for 30 min) inactivates SARS-CoV-2. Thus, in the event that donated human milk contains SARS-CoV-2 by transmission through the mammary gland or by contamination, this method of pasteurization renders milk safe for consumption and handling by care providers.

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector–based assay.

SARS-CoV-2 is a newly emerged betacoronavirus and the causative agent for the COVID-19 pandemic. Antibodies recognizing the viral spike protein are instrumental in natural and vaccine-induced immune responses to the pathogen and in clinical diagnostic and therapeutic applications. Unlike conventional immunoglobulins, the variable lymphocyte receptor antibodies of jawless vertebrates are structurally distinct, indicating that they may recognize different epitopes. Here we report the isolation of monoclonal variable lymphocyte receptor antibodies from immunized sea lamprey larvae that recognize the spike protein of SARS-CoV-2 but not of other coronaviruses. We further demonstrate that these monoclonal variable lymphocyte receptor antibodies can efficiently neutralize the virus and form the basis of a rapid, single step SARS-CoV-2 detection system. This study provides evidence for monoclonal variable lymphocyte receptor antibodies as unique biomedical research and potential clinical diagnostic reagents targeting SARS-CoV-2.

Continuously emerging SARS-CoV-2 Omicron subvariants pose a threat thwarting the effectiveness of approved COVID-19 vaccines. Especially, the protection breadth and degree of these vaccines against antigenically distant Omicron subvariants is unclear. Here, we report the immunogenicity and efficacy of a bivalent mRNA vaccine, PTX-COVID19-M1.2 (M1.2), which encodes native spike proteins from Wuhan-Hu-1 (D614G) and Omicron BA.2.12.1, in mouse and hamster models. Both primary series and booster vaccination using M1.2 elicited potent and broad nAbs against Wuhan-Hu-1 (D614G) and some Omicron subvariants. Strong spike-specific T cell responses against Wuhan-Hu-1 and Omicron subvariants, including JN.1, were also induced. Vaccination with M1.2 protected animals from Wuhan-Hu-1 and multiple Omicron subvariants challenges. Interestingly, protection against XBB.1.5 lung infection did not correlate with nAb levels. These results indicate that M1.2 generated a broadly protective immune response against antigenically distant Omicron subvariants, and spike-specific T cells probably contributed to the breadth of the protection.

Le recours aux dons de lait maternel pasteurisé est la norme de soins dans les hôpitaux pour les nourrissons ayant un très faible poids à la naissance, afin de faire le pont en attendant que les mères puissent allaiter leur enfant. Le but de cette étude était de vérifier si la pasteurisation à l’aide de la méthode de Holder (à 62,5°C pendant 30 min) serait suffisante pour inactiver le coronavirus du syndrome respiratoire aigu sévère 2 (SRAS-CoV-2) dans des échantillons de lait maternel provenant de donneuses. Nous avons inoculé avec le SRAS-CoV-2 des échantillons de lait congelés provenant de 10 donneuses de la Rogers Hixon Ontario Human Milk Bank (la banque de lait maternel de l’Ontario) pour atteindre une concentration finale de 1 × 107 DICT50/mL (50 % de la dose infectante de la culture de tissus par mL). Les échantillons ont été pasteurisés à l’aide de la méthode de Holder ou laissés à la température du laboratoire pendant 30 minutes, puis nous avons mis en culture des dilutions en série sur des cellules Vero E6 durant 5 jours. Nous avons utilisé des échantillons témoins dans cette étude, soit des échantillons de lait provenant des mêmes donneuses, auxquels le virus n’a pas été ajouté (échantillons pasteurisés et non pasteurisés), de même que des réplicats de cellules Vero E6 directement inoculées avec le SRAS-CoV-2. Nous rapportons ici les effets cytopathologiques en DICT50/mL. Nous n’avons détecté aucune activité cytopathologique dans l’ensemble des échantillons de lait contenant le SRAS-CoV-2 pasteurisés à l’aide de la méthode de Holder. Dans les échantillons contenant le SRASCoV-2 qui n’ont pas été pasteurisés, mais plutôt laissés à la température du laboratoire pendant 30 minutes, nous avons observé une réduction du titre infectieux d’environ 1 log. La pasteurisation du lait maternel à l’aide de la méthode de Holder (à 62,5°C pendant 30 min) inactive le SRAS-CoV-2. Ainsi, si du lait maternel provenant de donneuses contenait le virus à la suite d’une transmission par la glande mammaire ou d’une contamination, cette méthode de pasteurisation rendrait le lait sans danger pour la consommation par le nourrisson et la manipulation par les travailleurs de la santé.

CD38 is a multifunctional cell surface receptor expressed on multiple cell lineages of hematopoietic origin with high levels of expression on human plasma cells. Previously, we isolated the monoclonal variable lymphocyte receptor B (VLRB) MM3 antibody from the evolutionarily distant sea lamprey, which recognized the CD38 ectoenzyme exclusively on human plasma cells in a manner that correlated with CD38 enzymatic activity. The plasma cell-specific binding of VLRB MM3 contrasts with the broad pattern of expression of CD38-determined conventional antibodies specific for this antigen. In an effort to facilitate the application of this unique reagent in combination with conventional antibody panels, we explored a strategy to generate VLRB MM3 tetramers. The resulting reagent maintained the threshold-based recognition of CD38. Increased sensitivity achieved with VLRB MM3 tetramers also showed preferential recognition of germinal center centroblasts over centrocytes. VLRB MM3 tetramers thus provided a unique and versatile single-step staining reagent for the detection of human CD38 that is readily incorporated into multi-color flow cytometry panels.

The main barrier towards an HIV cure is the presence of latently HIV-infected cells that make up the viral reservoir which persists through antiretroviral therapy. Although there is some leaky protein transcription in these latent cells, they evade immune surveillance and through homeostatic proliferation, maintain the viral reservoir. To eradicate the viral reservoir, one must be able to target it through the use of a biomarker. In the first part of this thesis, sea lamprey were immunized with latently HIV-infected T cells to generate a library of lamprey antibodies. 1273 antibodies were isolated and screened against HIV infected and non-infected primary cells. 25 antibodies were found to be specific to HIV infected cells which are currently being classified further.Due to the COVID pandemic, HIV research was placed on ‘hold’ in order to develop SARS-CoV-2 neutralization assays and COVID vaccine development efforts. In chapter 3 we report the SARS-CoV-2 neutralization titers of human convalescent sera were consistent across all disease states except for severe COVID-19, which yielded significantly higher neutralization titers. Heat inactivation of human convalescent serum was shown to inactivate complement proteins, and the contribution of the complement system in SARS-CoV-2 neutralization was often over 50% and mainly driven through the classical pathway. In some cases, heat inactivation completely abolished neutralization levels to undetectable levels. This effect was also observed in COVID-19 vaccines and could be abolished in individuals who were being treated with anti-complement antibodies for other diseases.Safe and effective vaccines are needed to end the COVID-19 pandemic. In the final chapter we report the preclinical development of a lipid nanoparticle formulated SARS-CoV-2 mRNA vaccine. Tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Mouse immune sera elicited by PTX-COVID19-B vaccination neutralized SARS-CoV-2 variants of concern, including the Alpha, Beta, Gamma and Delta lineages. No adverse effects were induced by PTX-COVID19-B in either mice or hamsters. Based on these results, PTX-COVID19-B was authorized by Health Canada and had passed a phase 2 clinical trial.