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Development of an effective HIV-1 vaccine has been a great challenge faced by the research community. Recently a novel strategy targeting the viral protease cleavage sites (PCSs) has been tested and shown promising results. This T cell-based vaccine strategy depends on individuals expressing certain HLA class I molecules and since each population has unique distributions of HLA class I alleles, population coverage analysis is required to assess feasibility. Utilizing the validated CD8 T cell epitope data from previous studies we conducted coverage analysis of an HIV-1 vaccine targeting the sequences surrounding all 12-PCSs, Gag-PCSs, and Pol-PCSs. The population coverage, average epitope hit, and minimum number of epitopes recognized by 90% of the population (PC90) was compiled for 66 countries and 16 geographical regions using the web tool provided by “Immune Epitope Database and Analysis Resource”. Our analysis shows that the coverage for an HIV-1 vaccine targeting sequences surrounding all 12 PCSs, 5 PCSs in Gag or 6 PCSs in Pol can cover ~ 70% to ~ 100% of the populations analyzed. There was no statistical difference in population coverages for the majority of populations examined when comparing the CD8 T cell epitope sets (12-PCSs, Gag-PCSs, and Pol-PCSs). As expected, vaccines targeting more sequences will have more CD8 T cell epitopes, as the mean average epitope hit for the 12-PCSs, Gag-PCSs, and Pol-PCSs across all countries studied was 9.45, 4.76, and 4.74, respectively, and across all geographical regions was 9.76, 4.99, and 4.92, respectively. The average PC90 for the 12-PCSs, Gag-PCSs, and Pol-PCSs across all countries studied was 2.53, 1.31, and 1.41, respectively, and across all geographical regions was 2.24, 1.23, and 1.29, respectively. Thus, vaccines targeting sequences surrounding the HIV-1 PCSs can cover broad populations; however, whether targeting a subset of the PCSs is sufficient to prevent acquisition requires further preclinical investigation.

Background: We analyzed the prevalence of pre-antiretroviral therapy (ART) drug resistance mutations (DRMs) in a Kenyan population. We also examined whether host HLA class I genes influence the development of pre-ART DRMs. Methods: The HIV-1 proviral DNAs were amplified from blood samples of 266 ART-naïve women from the Pumwani Sex Worker cohort of Nairobi, Kenya using a nested PCR method. The amplified HIV genomes were sequenced using next-generation sequencing technology. The prevalence of pre-ART DRMs was investigated. Correlation studies were performed between HLA class I alleles and HIV-1 DRMs. Results: Ninety-eight percent of participants had at least one DRM, while 38% had at least one WHO surveillance DRM. M184I was the most prevalent clinically important variant, seen in 37% of participants. The DRMs conferring resistance to one or more integrase strand transfer inhibitors were also found in up to 10% of participants. Eighteen potentially relevant (p < 0.05) positive correlations were found between HLA class 1 alleles and HIV drug-resistant variants. Conclusions: High levels of HIV drug resistance were found in all classes of antiretroviral drugs included in the current first-line ART regimens in Africa. The development of DRMs may be influenced by host HLA class I-restricted immunity.

TILRR (Toll-like Interleukin-1 Receptor Regulator) is a modulator of many genes in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. It promotes the production of inflammatory mediators and the migration of immune cells. Recently, we showed that TILRR protein circulates in human blood. Thus, it could influence systemic inflammation. Systemic and mucosal inflammations increase the susceptibility to HIV infection. In this study, we analyzed the TILRR protein levels of the archived plasma samples of women enrolled in the Pumwani cohort to determine whether the plasma TILRR protein levels before seroconversion are correlated with differential risk of HIV seroconversion.TILRR protein of 941 archived HIV negative plasma samples from 390 women who were HIV negative at the cohort enrollment was quantified with an in-house developed multiplex bead array method. Proinflammatory cytokines/chemokines were measured using a 14-plex bead array method. Spearman rank correlation analysis was used to determine the correlation between plasma TILRR protein and proinflammatory cytokines/chemokines. Kaplan-Meier survival analysis was conducted to evaluate whether the median plasma TILRR protein levels correlate with increased risk of HIV seroconversion.The level of plasma TILRR protein was positively correlated with plasma IL-1β (rho: 0.2593, p0.0001), MCP-1 (rho: 0.2377, p0.0001), and IL-17A (rho: 0.1225, p=0.0216). Women with median plasma TILRR protein levels ≥100 ng/ml seroconverted significantly faster than women with plasma TILRR protein levels100 ng/ml (log-rank= 100.124, p0.0001; relative risk= 3.72 and odds ratio= 15.29). Furthermore, the factors causing genital inflammation, such as STIs (sexually transmitted infections), vaginal discharge, and genital ulcers were not statistically significantly different among women with different median plasma TILRR protein levels.The high plasma TILRR protein levels are highly correlated with several plasma proinflammatory cytokines/chemokines. High median plasma TILRR protein (≥100 ng/ml) strongly predicted an increased risk of HIV seroconversion. Reducing plasma TILRR protein levels may reduce the risk of HIV acquisition.The study was funded by an operating grant from the Canadian Institutes of Health Research (CIHR), operating grant-PA: CHVI Vaccine Discovery and Social Research (http://www.cihr-irsc.gc.ca/e/193.html), and National Microbiology Laboratory of Canada.

The macaque simian or simian/human immunodeficiency virus (SIV/SHIV) challenge model has been widely used to inform and guide human vaccine trials. Substantial advances have been made recently in the application of repeated-low-dose challenge (RLD) approach to assess SIV/SHIV vaccine efficacies (VE). Some candidate HIV vaccines have shown protective effects in preclinical studies using the macaque SIV/SHIV model but the model's true predictive value for screening potential HIV vaccine candidates needs to be evaluated further. Here, we review key parameters used in the RLD approach and discuss their relevance for evaluating VE to improve preclinical studies of candidate HIV vaccines.

COVID-19 has greatly affected public health and world economy. In this study, we analyzed 129 full-length genomes of SARS-CoV-2 viruses of a Canadian population during early phase of the pandemic. Phylogenetic analysis revealed three major paths of transmission of SARS-CoV-2 viruses into Canada. Twenty-one substitutions that have frequencies greater than 3% of viral population were identified. Analysis of these substitutions indicated that P1427I (ORF1b), Y1464C (ORF1b), and Q57H (ORF3a) might affect functions of the corresponding SARS-CoV-2 encoded proteins. Additionally, we found the evidence of positive selection on the ORF3a and codon 614 of Spike protein, suggesting the viral components responsible for host entry and activation of inflammation response were targeted by host immune responses. The study showed genomic variation and evolution of SARS-CoV-2 in a Canadian population. These information may help develop preventive strategies and be used for further study of SARS-CoV-2 pathogenesis and therapeutics development.

After over 3 decades of research, an effective anti-HIV vaccine remains elusive. The recently halted HVTN702 clinical trial not only further stresses the challenge to develop an effective HIV vaccine but also emphasizes that unconventional and novel vaccine strategies are urgently needed. Here, we report that a vaccine focusing the immune response on the sequences surrounding the 12 viral protease cleavage sites (PCSs) provided greater than 80% protection to Mauritian cynomolgus macaques against repeated intravaginal SIVmac251 challenges. The PCS-specific T cell responses correlated with vaccine efficacy. The PCS vaccine did not induce immune activation or inflammation known to be associated with increased susceptibility to HIV infection. Machine learning analyses revealed that the immune microenvironment generated by the PCS vaccine was predictive of vaccine efficacy. Our study demonstrates, for the first time to our knowledge, that a vaccine which targets only viral maturation, but lacks full-length Env and Gag immunogens, can prevent intravaginal infection in a stringent macaque/SIV challenge model. Targeting HIV maturation thus offers a potentially novel approach to developing an effective HIV vaccine.

Background:Human immunodeficiency virus-1 (HIV-1) mutates rapidly to escape host immune pressure. This results in the generation of positively selected mutations (PSM) throughout the viral genome. Escape mutations in Nef, one of the accessory proteins of HIV-1, which plays an important role in viral pathogenicity have previously been identified in several large cohort studies, but the evolution of PSMs overtime in various HIV-1 subtypes remains unknown.Methods:161 clade A1, 3093 clade B, 647 clade C and 115 clade D HIV-1 nef sequences were obtained from the HIV Database of Los Alamos National Laboratory and aligned using MEGA 6.0. The sequences from each clade were grouped based on the year of collection. Quasi analysis was used to identify PSMs and the number and locations of PSMs were compared among different subtypes.Results:PSMs for all four subtypes were distributed across the sequence of Nef, and conserved residues F90, W113, PxxPxR (a.a 72-77) remain unaltered overtime. The frequency of PSMs was stable among subtype B sequences but increased overtime for other subtypes. Phylogenetic analysis shows that sequences containing PSMs tend to cluster together at both inter and intra- subtype levels.Conclusion:Identification of PSMs and their changes overtime within various subtypes of HIV-1 is important in defining global viral evolutionary patterns that can provide insights for designing therapeutic strategies.

FREM1 (Fras-related extracellular matrix 1) and its splice variant TILRR (Toll-like interleukin-1 receptor regulator) have been identified as integral components of innate immune systems. The potential involvement of FREM1 in HIV-1 (human immunodeficiency virus 1) acquisition was suggested by a genome-wide SNP (single nucleotide polymorphism) analysis of HIV-1 resistant and susceptible sex workers enrolled in the Pumwani sex worker cohort (PSWC) in Nairobi, Kenya. The studies showed that the minor allele of a FREM1 SNP rs1552896 is highly enriched in the HIV-1 resistant female sex workers. Subsequent studies showed that FREM1 mRNA is highly expressed in tissues relevant to mucosal HIV-1 infection, including cervical epithelial tissues, and TILRR is a major modulator of many genes in the NF-κB signal transduction pathway. In this article, we review the role of FREM1 and TILRR in modulating inflammatory responses and inflammation, and how their influence on inflammatory responses of cervicovaginal tissue could enhance the risk of vaginal HIV-1 acquisition.

After over three decades of research, an effective anti-HIV vaccine remains elusive. Unconventional and novel vaccine strategies are needed. Here, we report that a vaccine focusing the immune response on the sequences surrounding the 12 viral protease cleavage sites (PCSs) provides greater than 80% protection of Mauritian cynomolgus macaques (MCMs) against repeated intravaginal SIVmac251 challenges. The PCS-specific T cell responses are correlated with vaccine efficacy. The PCS vaccine does not induce immune activation and inflammation known to be associated with increased susceptibility to HIV infection. Machine learning analyses revealed that the immune environment generated by the PCS vaccine predicts vaccine efficacy. Our study demonstrates for the first time that a novel vaccine which targets viral maturation, but lacks full Env and Gag proteins as immunogens, can prevent intravaginal infection in a highly stringent NHP/SIV challenge model. Targeting HIV maturation thus offers a novel approach to developing an effective HIV vaccine.One Sentence SummaryThe anti-PCS T cell responses and the immune environment induced by the novel PCS vaccine are key correlates of vaccine efficacy

Simian immunodeficiency virus (SIV) infection of Mauritian cynomolgus macaques (MCMs) is an increasingly important nonhuman primate model for HIV vaccine research. We previously reported that in MCMs anti-SIV antibodies can be naturally developed without exogenous infection or vaccination, and that a vaccine targeting SIV protease cleavage sites (PCS) can cross-induce antibodies to non-PCS SIV antigens. We speculate that this is potentially caused by the existence of endogenous SIV-like antigens. External stimuli (such as environmental factors and vaccination) may induce expression of endogenous SIV-like antigens to elicit these antibodies. Database and mass spectrometry analyses were conducted to search for such antigens. We identified endogenous SIV-like DNA sequences in cynomolgus macaque genome and non-PCS peptide homologous to SIV Env protein in PBMCs of a PCS-vaccinated monkey. Our preliminary insights suggest that endogenous SIV-like antigens may be one of the possible reasons for the natural and cross-inducible SIV antibodies in MCMs.

Identifying HCV drug resistance mutations (DRMs) is increasingly important as new direct acting antiviral therapies (DAA) become available. Tagged pooled pyrosequencing (TPP) was originally developed as cost-effective approach for detecting low abundance HIV DRMs. Using 127 HCV-positive samples from a Canadian injection drug user cohort, we demonstrated the suitability and efficiency of TPP for evaluating DRMs in HCV NS5B gene. At a mutation identification threshold of 1%, no nucleoside inhibitor DRMs were detected among these DAA naïve subjects. Clinical NS5B resistance to non-nucleoside inhibitors and interferon/ribavirin was predicted to be low within this cohort. S282T mutation, the primary mutation selected by sofosbuvir in vitro, was not identified while S282G/C/R variants were detected in 9 subjects. Further characterization on these new S282 variants using in silico molecular modeling implied their potential association with resistance. Combining TPP with in silico analysis detects NS5B polymorphisms that may explain differences in treatment outcomes.

Objective To demonstrate how a transvaginal natural orifice transluminal endoscopic surgery (NOTES) tubal reanastomosis is a novel route for tubal surgery. The surgical technique is a combination of traditional vaginal surgery with single-site surgical skills. Design The surgical technique is explained in a stepwise fashion with the use of surgical video footage. The video uses a surgical case to demonstrate the specific techniques necessary to perform a NOTES tubal reanastomosis. Setting Teaching university. Patient(s) A 42-year-old female G2P2 with a history of tubal ligation 11 years before presentation requesting a tubal recanalization. Intervention(s) Transvaginal NOTES tubal reanastomosis was initiated with a posterior colpotomy. A single-site gelport was placed. The fallopian tubes were hydrodissected, the blocked portion of each tube was removed, an epidural catheter was threaded through each lumen, and the two remaining segments of each tube were sutured together in an end-to-end fashion using single-site suturing skills. Main Outcome Measure(s) Transvaginal NOTES tubal reanastomosis as an alternative route for tubal reanastomosis. Result(s) The bilateral fallopian tubes were recanalized with bilateral tubal patency. This was confirmed 8 weeks postoperatively with a three-dimensional sonohystogram, which showed patency of the bilateral fallopian tubes. Conclusion(s) The current preferred technique for reversal of a tubal sterilization is to perform a minimally invasive surgery with an end-to-end anastomosis. This gives the patient a 60%–90% intrauterine pregnancy rate postoperatively. NOTES has the benefits of a fast recovery, no abdominal incisional pain, and an extremely cosmetic outcome. Current research has shown a 0%–3.1% range for the risk of pelvic infection in transvaginal NOTES if prophylactic antibiotics are administered during the surgery. The NOTES tubal reanastomosis combines the traditional vaginal surgery technique of creating a posterior colpotomy with single-site surgical skills like suturing and knot tying. The surgery is completed through a single transvaginal port without an abdominal incision. In the hands of a skilled minimally invasive surgeon, transvaginal NOTES tubal reanastomosis is a feasible and alternative route for this procedure.

The prevalence of drug resistance (DR) mutations in people with HIV-1 infection, particularly those with low-level viremia (LLV), supports the need to improve the sensitivity of amplification methods for HIV DR genotyping in order to optimize antiretroviral regimen and facilitate HIV-1 DR surveillance and relevant research. Here we report on a fully validated PCR-based protocol that achieves consistent amplification of the protease (PR) and reverse transcriptase (RT) regions of HIV-1 pol gene across many HIV-1 subtypes from LLV plasma samples. HIV-spiked plasma samples from the External Quality Assurance Program Oversight Laboratory (EQAPOL), covering various HIV-1 subtypes, as well as clinical specimens were used to optimize and validate the protocol. Our results demonstrate that this protocol has a broad HIV-1 subtype coverage and viral load span with high sensitivity and reproducibility. Moreover, the protocol is robust even when plasma sample volumes are limited, the HIV viral load is unknown, and/or the HIV subtype is undetermined. Thus, the protocol is applicable for the initial amplification of the HIV-1 PR and RT genes required for subsequent genotypic DR assays.

Cynomolgus macaques are an increasingly important nonhuman primate model for HIV vaccine research. SIV-free animals without pre-existing anti-SIV immune responses are generally needed to evaluate the effect of vaccine-induced immune responses against the vaccine epitopes. Here, in order to select such animals for vaccine studies, we screened 108 naïve female Mauritian cynomolgus macaques for natural (baseline) antibodies to SIV antigens using a Bio-Plex multiplex system. The antigens included twelve 20mer peptides overlapping the twelve SIV protease cleavage sites (-10/+10), respectively (PCS peptides), and three non-PCS Gag or Env peptides. Natural antibodies to SIV antigens were detected in subsets of monkeys. The antibody reactivity to SIV was further confirmed by Western blot using purified recombinant SIV Gag and Env proteins. As expected, the immunization of monkeys with PCS antigens elicited anti-PCS antibodies. However, unexpectedly, antibodies to non-PCS peptides were also induced, as shown by both Bio-Plex and Western blot analyses, while the non-PCS peptides do not share sequence homology with PCS peptides. The presence of natural and vaccine cross-inducible SIV antibodies in Mauritian cynomolgus macaques should be considered in animal selection, experimental design and result interpretation, for their best use in HIV vaccine research This work was supported by an NIH grant (R01AI111805), a CIHR/CHVI bridging grant and funding from National Microbiology Laboratory of Canada SI

Study Objective Transvaginal surgery is the most minimally invasive surgery for a gynecologic procedure, but it has the limitation of lack of exposure and limited surgical space when using traditional vaginal surgical instrumentation, such as in a hysterectomy for a uterus without descent or for a myomectomy. Transvaginal natural orifice transluminal endoscopic surgery (NOTES) offers similar benefits of traditional vaginal surgery but also expands the horizon of transvaginal surgery by allowing the surgeon to perform procedures that are typically limited to an abdominal approach. The advantages of NOTES may include no incisional pain as well as a better cosmetic outcome. These benefits help outweigh the obstacle of learning this novel approach. Our objective is to demonstrate the transvaginal NOTES technique as a combination of traditional vaginal surgical skill with single-site surgical skill. Design Stepwise demonstration of the transvaginal NOTES technique for myomectomy with narrated video footage (Canadian Task Force classification III). Setting Academic tertiary care hospital. Patient A 42-year-old woman. Interventions Transvaginal NOTES myomectomy with combined transvaginal surgical and single-site surgical skills. Measurements and Main Results A 42-year-old woman (gravida 2 para 2) with a preoperative transvaginal ultrasound diagnosis of a 6-cm left anterior myoma requested myoma removal with uterine preservation. She presented with a 2-year history of left pelvic pain and menorrhagia. The myoma was removed with minimal blood loss, and pathology revealed a necrotic myoma. The patient had resolution of her left-sided pelvic pain. Conclusions Combined with traditional transvaginal anterior colpotomy, single-site surgical skills allow the surgeon to access the entire abdomen and perform myomectomy through a transvaginal single port. Transvaginal NOTES myomectomy is not only possible but allows myomectomy to be performed with no abdominal incision.

Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. The complexity of cancer can be reduced to a small number of underlying principles like cancer hallmarks which could govern the transformation of normal cells to cancer. Besides, the growth and metastasis of cancer often relate to combined effects of long non-coding RNAs (lncRNAs). Here, we performed comprehensive analysis for lncRNA expression profiles and clinical data of six types of human cancer patients from The Cancer Genome Atlas (TCGA), and identified six risk pathways and twenty three lncRNAs. In addition, twenty three cancer risk lncRNAs which were closely related to the occurrence or development of cancer had a good classification performance for samples of testing datasets of six cancer datasets. More important, these lncRNAs were able to separate samples in the entire cancer dataset into high-risk group and low-risk group with significantly different overall survival (OS), which was further validated in ten validation datasets. In our study, the robust and effective cancer biomarkers were obtained from cancer datasets which had information of normal-tumor samples. Overall, our research can provide a new perspective for the further study of clinical diagnosis and treatment of cancer.

The first North American outbreak of highly pathogenic avian influenza (HPAI) involving a virus of Eurasian A/goose/Guangdong/1/1996 (H5N1) lineage began in the Fraser Valley of British Columbia, Canada in late November 2014. A total of 11 commercial and 1 non-commercial (backyard) operations were infected before the outbreak was terminated. Control measures included movement restrictions that were placed on a total of 404 individual premises, 150 of which were located within a 3 km radius of an infected premise(s) (IP). A complete epidemiological investigation revealed that the source of this HPAI H5N2 virus for 4 of the commercial IPs and the single non-commercial IP likely involved indirect contact with wild birds. Three IPs were associated with the movement of birds or service providers and localized/environmental spread was suspected as the source of infection for the remaining 4 IPs. Viral phylogenies, as determined by Bayesian Inference and Maximum Likelihood methods, were used to validate the epidemiologically inferred transmission network. The phylogenetic clustering of concatenated viral genomes and the median-joining phylogenetic network of the viruses supported, for the most part, the transmission network that was inferred by the epidemiologic analysis.

HIV mutates rapidly and infects CD4+ T cells, especially when they are activated. A vaccine targeting conserved, essential viral elements while limiting CD4+ T cell activation could be effective. Learning from natural immunity observed in a group of highly HIV-1 exposed seronegative Kenyan female sex workers, we are testing a novel candidate HIV vaccine targeting the 12 viral protease cleavage sites (PCSs) (the PCS vaccine), in comparison with a vaccine targeting full-length Gag and Env (the Gag/Env vaccine) in a Mauritian cynomolgus macaque/SIV model. In this study we evaluated these vaccines for induction of mucosal antibodies to SIV immunogens at the female genital tract. Bio-Plex and Western blot analyses of cervicovaginal lavage samples showed that both the PCS and Gag/Env vaccines can elicit mucosal IgG antibody responses to SIV immunogens. Significantly higher increase of anti-PCS antibodies was induced by the PCS vaccine than by the Gag/Env vaccine (p