Background: Bacterial vaginosis (BV) is associated with enhanced HIV risk, likely due to the induction of genital inflammation and associated epithelial barrier disruption. Lactobacillus crispatus is associated with immune quiescence and reduced HIV risk. We investigated the sustained impact of a L. crispatus-based live biotherapeutic (LACTIN-V) on proinflammatory cytokines, markers of epithelial disruption, and the vaginal microbiome, which was recently demonstrated to reduce BV recurrence (NCT02766023).

Methods: In this pilot study, soluble immune factor levels and the absolute abundance of key bacteria species were assayed for 48 participants who reported near-perfect adherence to assigned treatment during the phase 2b randomized double-blind placebo-controlled trial of LACTIN-V to prevent BV recurrence. All participants received topical metronidazole for 5-days and were then randomized 2:1 to LACTIN-V or matched placebo for 10 weeks. Vaginal swabs were collected before and after metronidazole therapy, and then at 4-, 8-, 12-, and 24-weeks. Cytokines were assayed using the MSD multiplex platform and the absolute abundance of bacteria species was measured with qPCR.

Results: Absolute abundance of L. crispatus was higher in the LACTIN-V arm compared to placebo at 12 weeks (p<0.001) and this increase was sustained for at least 3 months after product cessation (p=0.013). These sustained increases in L. crispatus abundance at 24 weeks correlated with reductions in vaginal concentrations of the prototypic inflammatory cytokine IL-1a (r=-0.2973, p=0.0402), the epithelial damage biomarker sE-cad (r=-0.4860, p=0.0005), and the BV-associated bacterial genus Prevotella (r=-0.4341, p=0.0021).

Conclusions: Treatment with LACTIN-V following standard antibiotic treatment enhanced L. crispatus colonization for at least 3 months after product cessation; these increases were associated with reduced inflammation and enhanced epithelial integrity, potentially due to the exclusion of BV-associated bacteria. Given the links between genital inflammation, epithelial disruption and HIV acquisition, LACTIN-V may represent a novel strategy to reduce HIV risk among women.

De nombreuses populations cellulaires sont dérégulées lors de l’infection par le VIH. Parmi celles-ci, on y retrouve les lymphocytes B de la zone marginale (MZ). Chez des individus sains, les populations de lymphocytes B innés de la zone marginale sont essentielles pour la formation des centres germinatifs et elles sont également capables de s’activer de façon T-indépendante et ainsi sécréter de nombreuses immunoglobulines polyréactives. De plus, nous avons récemment démontré que les précurseurs des MZ (MZp) ont un fort potentiel régulateur (Breg). Nous avions précédemment démontré que dans un contexte d’infection au VIH, l’excès du B-cell activating factor (BAFF) est concomitant avec l’augmentation de la fréquence des MZp. Afin de mieux comprendre cette dérégulation, une analyse du transcriptome par RNAseq a été effectuée sur des MZ et MZp du sang d’individus VIH+. L’expression des TLR7 et 10 sont augmentés, ce qui est compatible avec une signature interféron (IFN) chez ces cellules. Aussi, l’expression des marqueurs T-bet et CD11c, qui sont associés aux « age associated B cells » impliquées dans certains contextes auto-immuns et inflammatoires, est plus importante que celle des MZ et MZp provenant d’individus sains. Ces deux marqueurs sont également augmentés suite à une activation des cellules via TLR7 ou par une réponse IFN. De plus, l’expression de certains marqueurs associés à l’épuisement ou à l’activation du BCR, tels que CD85j, CD22, FCRL5 et CD72 est aussi plus importante chez les cellules MZ et MZp d’individus VIH+. Ensemble, ces résultats suggèrent que les MZ et MZp sont hyperactivées, épuisées et qu’elles présentent un profil similaire aux « age associated B cells » aussi retrouvées dans plusieurs infections inflammatoires chroniques.

Over the past year, SARS-CoV-2 has infected over 90 million people globally and resulted in nearly 2 million deaths. While novel vaccines have been developed and deployed rapidly to counteract this virus, the durabilty of antibody responses elicited by these vaccines is still uncertain. New research tools are thus needed to evaluate humoral immune responses against SARS-CoV-2 infection and vaccines.

We designed a Luminex-based assay to simultaneously quantify antibodies targeting the receptor binding domain (RBD) of SARS-CoV-2 and five related human coronaviruses (SARS, HKU1, NL63, OC43, 229E). This assay can assess the ability of antibodies to block RBD interactions with human angiotensin converting enzyme (ACE-2) receptor. Briefly, each RBD target was coupled to a distinct xMAP bead and validated using commercial antibodies. Beads were mixed with patient sera alone or sera supplemented with recombinant ACE-2. Bound IgG or IgM antibodies were quantified using a Luminex instrument. Reduced antibody binding observed in the presence of ACE-2 provides a surrogate measure of potential neutralizing activity in sera.

In a pilot study of sera from 10 SARS-CoV-2 patients, including 5 that displayed virus neutralizing activity, our results correlated with prior data collected using more traditional ELISA. Sera from virus neutralizers displayed higher amounts of SARS-CoV-2 RBD binding IgG antibodies (p=0.03) and were better able to compete with ACE-2 (p<0.01), both of which are consistent with better neutralization activity.

In summary, we developed a higher-throughput multiplex method to quantify and characterize antibody responses elicited following SARS-CoV-2 infection. This new research tool should enhance our ability to evaluate the efficacy and durability of current COVID-19 vaccines, and provide a new platform to test future vaccine candidates and antibody-based therapeutics.

Despite the advent of safe and effective vaccines, the scientific community continues to work to understand the immune pathology that drives severe COVID-19 disease and to develop effective treatments against SARS-CoV-2. Thus, it is imperative to develop useful animal models that sufficiently recapitulate the immune-mediated lung pathology observed during SARS-CoV-2 infection and to rapidly evaluate candidate therapeutics. We modified our bone marrow, liver, thymus humanized C57BL/6-Rag2-/-gammac-/-CD47-/- triple knock-out (TKO-BLT) mouse model to support two subcutaneously implanted autologous human lung organoids to produce the TKO-BLT-Lung (TKO-BLT-L) mouse model of SARS-CoV-2 infection. Twelve weeks post-humanization the TKO-BLT-L mouse was engrafted with high levels of human immune cells (>2x105 human CD45+ cells/ml of blood). Within TKO-BLT-L mice, lung organoids grew to ~10mm in diameter and exhibited significant vascularization as assessed by ultrasound imaging. Histologic sections of human lung implants showed the presence of airways, ciliated epithelium, alveolar structures, cartilage and associated blood vessels. The TKO-BLT-L mice were successfully infected with SARS-CoV-2 virus via direct injection into the human lung organoid and supported detectable viral infection over a 10-day period based on qPCR and plaque assay. Immunohistochemical staining of lung organoids showed the localization of resident human alveolar macrophages pre-infection and immune infiltration and associated lung tissue damage upon infection with SARS-CoV-2. The TKO-BLT-L mouse model will allow the study of the immune mediated lung pathology observed during COVID-19 disease and the evaluation of therapeutic candidates in the context of genuine human lung tissue and a functional human immune system.

Background. At least three approved vaccines are now available to reduce the spread of the COVID-19 pandemic. However, the quality and quantity of protective immune mechanisms induced following infection or vaccination remains unknown, particularly in the upper respiratory tract (URT), the primary site of viral entry. We developed in an ex vivo flow cytometry-based assay to enumerate and profile immune cells isolated from specific nasopharyngeal (NP) swabs used for SARS-CoV-2 diagnostic testing amongst healthy volunteers.

Methods. We pilot-tested several types of NP swabs to gauge optimal immune cell recovery. Cells were obtained from swabs via mechanical means, washed, and stained using a pre-titrated panel of fluorescently labelled monoclonal antibodies. Data were acquired on a BD LSRFortessaTM cytometer and analyzed using Flow Jo V10.

Results. Of the NP swabs we tested, only the BD paediatric swab yielded CD45+ as opposed to CD326+ epithelial cells. In BD pediatric swabs (n=8), a median of 3,082 (IQR: 2,351-6,168) CD45+ cells were recovered per swab, ~80% of which were CD3+ T cells. Using CD69 and CD103 as markers of tissue resident memory (Trm) T cells, virtually all CD8+ (>90%) and variable proportions of CD4+ T cells were Trm (10-80%). Our panel also captures the activation status of these cells (CD38, HLA-DR, perforin, Ki-67), and CD4 phenotypes, such as Th17 (CD161, CCR6), Tfh (CXCR5, PD1) and antigen-specificity (CD40L, OX40). Efforts are currently underway to apply these methods to characterize nasal T cells pre- and post-COVID-19 vaccination.

Conclusions. Nasal T cell studies are feasible due to successful isolation of T cells from certain types of NP swabs. A high proportion of these T cells appear to be tissue resident. Examining protective immune responses induced in the URT following immunization with COVID-19 mRNA vaccines may be critical to understanding the nature and localization of this protection.

Background. Residents of long-term care (LTC) facilities are at significantly higher risk of severe COVID-19. Two-dose mRNA vaccines greatly reduce risk of hospitalization and mortality; however, limited vaccine availability has led many Canadian provinces to delay second doses. While this delay is supported by reductions in COVID-19 outbreaks in LTC facilities following initial vaccine rollout, few data are available on immunogenicity of a single mRNA vaccine dose among the elderly.

Methods. We studied 12 LTC residents (median age, 82 years) and 18 healthcare workers (HCW; median age, 36 years). Plasma/sera were collected pre-vaccine and one month following the first dose of BNT162b2 vaccine. Total antibody responses to SARS-CoV-2 nucleocapsid (N) and spike protein receptor binding domain (RBD) were assessed using commercial immunoassays (Roche). Luminex-based ELISA was used to quantify IgG and IgM to RBD and to determine the ability of antibodies to compete ACE2 receptor binding to RBD. Neutralizing antibody responses were assessed using pseudovirus and live SARS-CoV-2.

Results. At baseline, all participants were negative for anti-N and anti-RBD antibodies, consistent with no prior SARS-CoV-2 infection. After one vaccine dose, binding antibody responses against RBD were ~4-fold lower in residents compared to HCW (p<0.001); one resident mounted no response above background. Consistent with this, antibody competition with ACE2 binding was 3-fold lower in residents compared to HCW (p=0.01) and pseudovirus neutralizing activity was 2-fold lower (p=0.003). Finally, while six HCW (33%) displayed marginal ability to neutralize SARS-CoV-2 infection at a 1:20 dilution, only one resident did (8%) (p=0.19).

Conclusions. The implications of our findings for SARS-CoV-2 transmission and COVID-19 outcomes are unclear, but the observation that residents of LTC facilities display significantly reduced humoral immune reactivity after one dose of mRNA vaccine suggests that the interval between the first and second dose should not be extended in elderly individuals.