Development of a Model of Tubulointerstitial Fibrosis Using Transient Unilateral Renal Ischemia and Delayed Contralateral Nephrectomy in Domesticated Cats.

Tubulointerstitial fibrosis is a classic histologic feature of chronic kidney disease (CKD) in cats and a final common pathway toward end-stage renal disease. Domesticated cats have been used in models of ischemia-induced renal fibrosis. The objective of this study was to evaluate the performance of 2 variations of a transient unilateral renal ischemia and delayed contralateral nephrectomy model of tubulointerstitial fibrosis in cats. Purpose-bred, young adult, domesticated cats underwent 90 min of surgically induced ischemia to the right kidney followed by delayed contralateral nephrectomy performed 21 d (RI-CN21d group; n = 10) or 90 d postischemia (RI-CN90d group; n = 12). Control cats underwent sham surgery followed by left nephrectomy 21 d after (sham-CN group; n = 3). Renal functional parameters, including glomerular filtration rate and serum creatinine concentration, were evaluated before and after surgeries. The right kidneys were harvested 120 d postischemia/sham. Renal histology with lesion scoring and histomorphometry for quantification of smooth muscle actin immunolabeling and collagen staining were performed on harvested kidneys. Severe acute kidney injury prompted euthanasia after left nephrectomy in 5/10 (50.0%), 2/12 (16.7%), and 0/3 (0%) of cats in the RI-CN21d, RI-CN90d, and sham-CN groups, respectively. A significant decrease in glomerular filtration rate by day 120, relative to baseline, occurred in cats in the RI-CN21d group (P < 0.001) and RI-CN90d group (P < 0.001) but not the sham-CN group (P = 0.76). All but one cat in the ischemia groups were azotemic at the study end. Kidneys subjected to ischemia had higher interstitial inflammation, tubular atrophy, and fibrosis scores compared with sham-operated kidneys. There were significant increases in smooth muscle actin immunolabeling and collagen staining in these kidneys, relative to the contralateral kidneys. In summary, 90 min of unilateral renal ischemia and delayed contralateral nephrectomy induced histologic and biochemical changes consistent with CKD in cats. A 90-d period between ischemia and nephrectomy resulted in improved survivability of the model.

In vitro generated antibodies guide thermostable ADDomer nanoparticle design for nasal vaccination and passive immunization against SARS-CoV-2.

Background: Due to COVID-19, pandemic preparedness emerges as a key imperative, necessitating new approaches to accelerate development of reagents against infectious pathogens. Methods: Here, we developed an integrated approach combining synthetic, computational and structural methods with in vitro antibody selection and in vivo immunization to design, produce and validate nature-inspired nanoparticle-based reagents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Results: Our approach resulted in two innovations: (i) a thermostable nasal vaccine called ADDoCoV, displaying multiple copies of a SARS-CoV-2 receptor binding motif derived epitope and (ii) a multivalent nanoparticle superbinder, called Gigabody, against SARS-CoV-2 including immune-evasive variants of concern (VOCs). In vitro generated neutralizing nanobodies and electron cryo-microscopy established authenticity and accessibility of epitopes displayed by ADDoCoV. Gigabody comprising multimerized nanobodies prevented SARS-CoV-2 virion attachment with picomolar EC50. Vaccinating mice resulted in antibodies cross-reacting with VOCs including Delta and Omicron. Conclusion: Our study elucidates Adenovirus-derived dodecamer (ADDomer)-based nanoparticles for use in active and passive immunization and provides a blueprint for crafting reagents to combat respiratory viral infections.

Utilization of the En Route Aeromedical Patient Movement Form by Critical Care Air Transport Teams.

INTRODUCTION: Understanding usage patterns of current paper-based documentation can inform the development of electronic documentation forms for en route care. The primary objective was to analyze the frequency of use of each field within the 3899 L Patient Movement Record documented by en route Critical Care Air Transport Teams. Secondary objectives were to identify rarely utilized form fields and to analyze the proportion of verifiable major events documented within the 3899 L form. MATERIALS AND METHODS: We performed a retrospective review of 3899 L patient movement records for patients transported via Critical Care Air Transport Teams from January 2019 to December 2019. Scanned 3899 L forms were manually transcribed into a Microsoft Access database for evaluation and analysis. Proportions were calculated for completed fields. Major vital sign event frequency was compared for checkbox fields versus the vital sign flow sheet for each patient. We performed descriptive analyses for the proportion of charts with completed documentation in each evaluated field and the proportion of flow sheet events documented in major event fields. RESULTS: We analyzed 130 records. Fourteen of 18 (77.8%) demographic fields had a 75% or greater completion ratio. Sections with the largest proportion of rarely or never utilized fields (<1.5% completed) were procedures (77.8% of fields) and major events (63.9% of fields). Major event checkboxes had low sensitivity for documented events in the flow sheet: Change in heart rate greater than 20% (1 of 28 patients); increase in the fraction of inspired oxygen requirement of greater than 10% (6 of 23 patients); decrease in mean arterial pressure of greater than 20% (1 in 12 patients); and temperature less than 35.6°C (1 in 13 patients). CONCLUSIONS: Many of the current 3899 L fields are highly utilized, but some 3899 L sections contain high proportions of rarely utilized fields. Major event checkboxes did not consistently capture events documented within the in-flight vital sign flow sheet.

The Ability of Military Critical Care Air Transport Members to Visually Estimate Percent Systolic Pressure Variation.

INTRODUCTION: Inappropriate fluid management during patient transport may lead to casualty morbidity. Percent systolic pressure variation (%SPV) is one of several technologies that perform a dynamic assessment of fluid responsiveness (FT-DYN). Trained anesthesia providers can visually estimate and use %SPV to limit the incidence of erroneous volume management decisions to 1-4%. However, the accuracy of visually estimated %SPV by other specialties is unknown. The aim of this article is to determine the accuracy of estimated %SPV and the incidence of erroneous volume management decisions for Critical Care Air Transport (CCAT) team members before and after training to visually estimate and utilize %SPV. MATERIAL AND METHODS: In one sitting, CCAT team providers received didactics defining %SPV and indicators of fluid responsiveness and treatment with %SPV ≤7 and ≥14.5 defining a fluid nonresponsive and responsive patient, respectively; they were then shown ten 45-second training arterial waveforms on a simulated Propaq M portable monitor's screen. Study subjects were asked to visually estimate %SPV for each arterial waveform and queried whether they would treat with a fluid bolus. After each training simulation, they were told the true %SPV. Seven days post-training, the subjects were shown a different set of ten 45-second testing simulations and asked to estimate %SPV and choose to treat, or not. Nonparametric limits of agreement for differences between true and estimated %SPV were analyzed using Bland-Altman graphs. In addition, three errors were defined: (1) %SPV visual estimate errors that would label a volume responsive patient as nonresponsive, or vice versa; (2) incorrect treatment decisions based on estimated %SPV (algorithm application errors); and (3) incorrect treatment decisions based on true %SPV (clinically significant treatment errors). For the training and testing simulations, these error rates were compared between, and within, provider groups. RESULTS: Sixty-one physicians (MDs), 64 registered nurses (RNs), and 53 respiratory technicians (RTs) participated in the study. For testing simulations, the incidence and 95% CI for %SPV estimate errors with sufficient magnitude to result in a treatment error were 1.4% (0.5%, 3.2%), 1.6% (0.6%, 3.4%), and 4.1% (2.2%, 6.9%) for MDs, RNs, and RTs, respectively. However, clinically significant treatment errors were statistically more common for all provider types, occurring at a rate of 7%, 10%, and 23% (all P < .05). Finally, students did not show clinically relevant reductions in their errors between training and testing simulations. CONCLUSIONS: Although most practitioners correctly visually estimated %SPV and all students completed the training in interpreting and applying %SPV, all groups persisted in making clinically significant treatment errors with moderate to high frequency. This suggests that the treatment errors were more often driven by misapplying FT-DYN algorithms rather than by inaccurate visual estimation of %SPV. Furthermore, these errors were not responsive to training, suggesting that a decision-making cognitive aid may improve CCAT teams' ability to apply FT-DYN technologies.

Understanding COVID-19 vaccine hesitancy in health care professionals in Central and West Asia: lessons for future emergency mass vaccination campaigns.

A Quick Assessment of Vaccine Hesitancy approach was developed to collect population insights on vaccination hesitancy for low resource environments. Insights into COVID-19 vaccine hesitancy were collected through online webinars with heads of healthcare departments and anonymized online surveys of healthcare managers (HCM) and primary healthcare workers (HCW) in four countries in Central and West Asia (Armenia, Georgia, Tajikistan, and Kyrgyzstan) between 28 February 2022 and 29 March 2022. From the responses to the survey some key themes identified that underpinned in vaccine hesitancy across the region were perceived understanding of vaccine efficacy, conflict with individual religious beliefs, concerns for side effects, and the relatively rapid development of the vaccine and that improving communications strategies to address these concerns would be critical in combatting vaccine hesitancy through any future public health emergencies.

Vaccine research and development capacity in Central and West Asia: A path toward sustainable vaccine R&D programs.

The ability to support a comprehensive vaccine research and development (R&D) portfolio from a health security perspective has taken on enhanced significance over the past 3 years whereby countries that had existing vaccine R&D infrastructure (G7, Russia and China) have been at the forefront of global efforts to combat COVID-19. Few countries outside of these key players have the infrastructure necessary to develop national vaccine programs, though this is beginning to change with investment across many low- and middle-income countries. These same opportunities exist for countries in Central and West Asia, and in this perspective, we highlight the existing infrastructure and expertise across seven countries (Armenia, Azerbaijan, Georgia, Kazakhstan, Kyrgyzstan, Tajikistan, and Uzbekistan) and propose opportunities for enhanced collaboration along with a bold proposal for establishing a new-build, regional vaccine translational research institute to facilitate the development of a robust, regional vaccine R&D environment to combat existing and future health challenges.

Descriptive Analysis of Intratheater Critical Care Air Transport Team Patient Movements During Troop Drawdown: Afghanistan (2017-2019).

BACKGROUND: The majority of critical care air transport (CCAT) flights are regulated, meaning that a theater-validating flight surgeon has confirmed that the patient is medically cleared for flight and that evacuation is appropriate. If the conditions on the ground do not allow for this process, the flight is unregulated. Published data are limited regarding CCAT unregulated missions to include the period of troop drawdown at the end of the Afghanistan conflict. The objective of our study was to characterize the unregulated missions within Afghanistan during troop drawdown and compare them to regulated missions during the same timeframe. STUDY DESIGN: We performed a retrospective review of all CCAT medical records of patients transported via CCAT within Afghanistan between January 2017 and December 2019. We abstracted data from the records, including mission characteristics, patient demographics, injury descriptors, preflight military treatment facility procedures, CCAT procedures, in-flight CCAT treatments, in-flight events, and equipment issues. Following descriptive and comparative analysis, a Cochran-Armitage test was performed to evaluate the statistical significance of the trend in categorical data over time. Multivariable regression was used to assess the association between vasopressors and preflight massive transfusions, preflight surgical procedures, injury patterns, and age. RESULTS: We reviewed 147 records of patients transported via CCAT: 68 patients were transported in a regulated fashion and 79 on an unregulated flight. The number of patients evacuated increased year-over-year (n = 22 in 2017, n = 57 in 2018, and n = 68 in 2019, P < .001), and the percentage of missions that were unregulated grew geometrically (14%, n = 3 in 2017; 37%, n = 21 in 2018; and 81%, n = 55 in 2019, P < .001). During the time studied, CCAT teams were being used more to decompress forward surgical teams (FST) and, therefore, they were transporting patients just hours following initial damage control surgery in an unregulated fashion. In 2 instances, CCAT decompressed an FST following a mass casualty, during which aeromedical evacuation (AE) crews assisted with patient care. For the regulated missions, the treatments that were statistically more common were intravenous fluids, propofol, norepinephrine, any vasopressors, and bicarbonate. During unregulated missions, the statistically more common treatments were ketamine, fentanyl, and 3% saline. Additional analysis of the mechanically ventilated patient subgroup revealed that vasopressors were used twice as often on regulated (38%) vs. unregulated (13%) flights. Multivariable regression analysis demonstrated that traumatic brain injury (TBI) was the only significant predictor of in-flight vasopressor use (odds ratio = 3.53, confidence interval [1.22, 10.22], P = .02). CONCLUSION: During the troop drawdown in Afghanistan, the number of unregulated missions increased geometrically because the medical footprint was decreasing. During unregulated missions, CCAT providers used ketamine more frequently, consistent with Tactical Combat Casualty Care guidelines. In addition, TBI was the only predictor of vasopressor use and may reflect an attempt to adhere to unmonitored TBI clinical guidelines. Interoperability between CCAT and AE teams is critical to meet mass casualty needs in unregulated mission environments and highlights a need for joint training. It remains imperative to evaluate changes in mission requirements to inform en route combat casualty care training.

Exposure to common infections may shape basal immunity and potentially HIV-1 acquisition amongst a high-risk population in Coastal Kenya.

Introduction: The impact of exposure to endemic infections on basal immunity and susceptibility to HIV-1 acquisition remains uncertain. We hypothesized that exposure to infections such as cytomegalovirus (CMV), malaria and sexually transmitted infections (STIs) in high-risk individuals may modulate immunity and subsequently increase susceptibility to HIV-1 acquisition. Methods: A case-control study nested in an HIV-1 negative high-risk cohort from Coastal Kenya was used. Cases were defined as volunteers who tested HIV-1 positive during follow-up and had a plasma sample collected 3 ± 2 months prior to the estimated date of HIV-1 infection. Controls were individuals who remained HIV-1 negative during the follow-up and were matched 2:1 to cases by sex, age, risk group and follow-up time. STI screening was performed using microscopic and serologic tests. HIV-1 pre-infection plasma samples were used to determined exposure to CMV and malaria using enzyme-linked immunosorbent assays and to quantify forty-one cytokines and soluble factors using multiplexing assays. Multiplexing data were analyzed using principal component analysis. Associations between cytokines and soluble factors with subsequent HIV-1 acquisition were determined using conditional logistic regression models. Results and discussion: Overall, samples from 47 cases and 94 controls were analyzed. While exposure to malaria (p=0.675) and CMV (p=0.470) were not associated with HIV-1 acquisition, exposure to STIs was (48% [95% CI, 33.3 - 63] vs. 26% [95% CI, 17.3 - 35.9]. Ten analytes were significantly altered in cases compared to controls and were clustered into four principal components: PC1 (VEGF, MIP-1ß, VEGF-C and IL-4), PC2 (MCP-1, IL-2 and IL-12p70), PC3 (VEGF-D) and PC4 (Eotaxin-3). PC1, which is suggestive of a Th2-modulatory pathway, was significantly associated with HIV-1 acquisition after controlling for STIs (adjusted odds ratio, (95% CI), p-value: 1.51 [1.14 - 2.00], p=0.004). Elevation of Th2-associated pathways may dampen responses involved in viral immunity, leading to enhanced susceptibility to HIV-1 acquisition. Immunomodulatory interventions aimed at inhibiting activation of Th2-associated pathways may be an additional strategy to STI control for HIV-1 prevention and may reduce dampening of immune responses to vaccination.

Assessment of a diverse panel of transmitted/founder HIV-1 infectious molecular clones in a luciferase based CD8 T-cell mediated viral inhibition assay.

Introduction: Immunological protection against human immunodeficiency virus-1 (HIV-1) infection is likely to require both humoral and cell-mediated immune responses, the latter involving cytotoxic CD8 T-cells. Characterisation of CD8 T-cell mediated direct anti-viral activity would provide understanding of potential correlates of immune protection and identification of critical epitopes associated with HIV-1 control. Methods: The present report describes a functional viral inhibition assay (VIA) to assess CD8 T-cell-mediated inhibition of replication of a large and diverse panel of 45 HIV-1 infectious molecular clones (IMC) engineered with a Renilla reniformis luciferase reporter gene (LucR), referred to as IMC-LucR. HIV-1 IMC replication in CD4 T-cells and CD8 T-cell mediated inhibition was characterised in both ART naive subjects living with HIV-1 covering a broad human leukocyte antigen (HLA) distribution and compared with uninfected subjects. Results & discussion: CD4 and CD8 T-cell lines were established from subjects vaccinated with a candidate HIV-1 vaccine and provided standard positive controls for both assay quality control and facilitating training and technology transfer. The assay was successfully established across 3 clinical research centres in Kenya, Uganda and the United Kingdom and shown to be reproducible. This IMC-LucR VIA enables characterisation of functional CD8 T-cell responses providing a tool for rational T-cell immunogen design of HIV-1 vaccine candidates and evaluation of vaccine-induced T-cell responses in HIV-1 clinical trials.

Optimization and validation of an ELISA assay for the determination of antibody responses to CN54gp140 and AIDSVAX BE for use in the Phase IIb PrEPVacc vaccine trial.

PrEPVacc is an international, multi-centre, double-blind vaccine study comparing experimental combination vaccine regimens including DNA/AIDSVAX BE and DNA/CN54gp140 with placebo control. Simultaneously, daily oral PrEP is compared for efficacy against daily Truvada in the context of the current PrEP availability situation at the study sites. An important clinical trial outcome is the accurate measurement of in vivo antibody titer induced through vaccination. Here we report the validation of two ELISAs for CN54gp140 and AIDSVAX BE at Uganda Virus Research Institute that demonstrates precision, specificity, and robustness for assessing the reciprocal antibody end point titer in human serum. This is a critical endpoint for determining whether vaccination can provide any protection against HIV in populations at risk of acquiring HIV.

Infection with HIV-1 subtype D among acutely infected Ugandans is associated with higher median concentration of cytokines compared to subtype A.

Objective: The observation that HIV-1 subtype D progresses faster to disease than subtype A prompted us to examine cytokine levels early after infection within the predominant viral subtypes that circulate in Uganda and address the following research questions: (1) Do cytokine levels vary between subtypes A1 and D? (2) Do cytokine profiles correlate with disease outcomes? Methods: To address these questions, HIV-1 subtypes were determined by population sequencing of the HIV-1 pol gene and 37 plasma cytokine concentrations were evaluated using V-Plex kits on Meso Scale Discovery platform in 65 recent sero-converters. Results: HIV-1 subtype D (pol) infections exhibited significantly higher median plasma concentrations of IL-5, IL-16, IL-1α, IL-7, IL-17A, CCL11 (Eotaxin-1), CXCL10 (IP-10), CCL13 (MCP-4) and VEGF-D compared to subtype A1 (pol) infections. We also found that IL-12/23p40 and IL-1α were associated with faster CD4+T cell count decline, while bFGF was associated with maintenance of CD4+ counts above 350 cells/microliter. Conclusion: Our results suggest that increased production of cytokines in early HIV infection may trigger a disruption of the immune environment and contribute to pathogenic mechanisms underlying the accelerated disease progression seen in individuals infected with HIV-1 subtype D in Uganda.

An assessment of marine, estuarine, and riverine habitat vulnerability to climate change in the Northeast U.S.

Climate change is impacting the function and distribution of habitats used by marine, coastal, and diadromous species. These impacts often exacerbate the anthropogenic stressors that habitats face, particularly in the coastal environment. We conducted a climate vulnerability assessment of 52 marine, estuarine, and riverine habitats in the Northeast U.S. to develop an ecosystem-scale understanding of the impact of climate change on these habitats. The trait-based assessment considers the overall vulnerability of a habitat to climate change to be a function of two main components, sensitivity and exposure, and relies on a process of expert elicitation. The climate vulnerability ranks ranged from low to very high, with living habitats identified as the most vulnerable. Over half of the habitats examined in this study are expected to be impacted negatively by climate change, while four habitats are expected to have positive effects. Coastal habitats were also identified as highly vulnerable, in part due to the influence of non-climate anthropogenic stressors. The results of this assessment provide regional managers and scientists with a tool to inform habitat conservation, restoration, and research priorities, fisheries and protected species management, and coastal and ocean planning.

Breadth of CD8 T-cell mediated inhibition of replication of diverse HIV-1 transmitted-founder isolates correlates with the breadth of recognition within a comprehensive HIV-1 Gag, Nef, Env and Pol potential T-cell epitope (PTE) peptide set.

Full characterisation of functional HIV-1-specific T-cell responses, including identification of recognised epitopes linked with functional antiviral responses, would aid development of effective vaccines but is hampered by HIV-1 sequence diversity. Typical approaches to identify T-cell epitopes utilising extensive peptide sets require subjects' cell numbers that exceed feasible sample volumes. To address this, CD8 T-cells were polyclonally expanded from PBMC from 13 anti-retroviral naïve subjects living with HIV using CD3/CD4 bi-specific antibody. Assessment of recognition of individual peptides within a set of 1408 HIV-1 Gag, Nef, Pol and Env potential T-cell epitope peptides was achieved by sequential IFNγ ELISpot assays using peptides pooled in 3-D matrices followed by confirmation with single peptides. A Renilla reniformis luciferase viral inhibition assay assessed CD8 T-cell-mediated inhibition of replication of a cross-clade panel of 10 HIV-1 isolates, including 9 transmitted-founder isolates. Polyclonal expansion from one frozen PBMC vial provided sufficient CD8 T-cells for both ELISpot steps in 12 of 13 subjects. A median of 33 peptides in 16 epitope regions were recognised including peptides located in previously characterised HIV-1 epitope-rich regions. There was no significant difference between ELISpot magnitudes for in vitro expanded CD8 T-cells and CD8 T-cells directly isolated from PBMCs. CD8 T-cells from all subjects inhibited a median of 7 HIV-1 isolates (range 4 to 10). The breadth of CD8 T-cell mediated HIV-1 inhibition was significantly positively correlated with CD8 T-cell breadth of peptide recognition. Polyclonal CD8 T-cell expansion allowed identification of HIV-1 isolates inhibited and peptides recognised within a large peptide set spanning the major HIV-1 proteins. This approach overcomes limitations associated with obtaining sufficient cell numbers to fully characterise HIV-1-specific CD8 T-cell responses by different functional readouts within the context of extreme HIV-1 diversity. Such an approach will have useful applications in clinical development for HIV-1 and other diseases.

Increased Frequency of Inter-Subtype HIV-1 Recombinants Identified by Near Full-Length Virus Sequencing in Rwandan Acute Transmission Cohorts.

Most studies of HIV-1 transmission have focused on subtypes B and C. In this study, we determined the genomic sequences of the transmitted founder (TF) viruses from acutely infected individuals enrolled between 2005 and 2011 into IAVI protocol C in Rwanda and have compared these isolates to viruses from more recent (2016-2019) acute/early infections in three at risk populations - MSM, high risk women (HRW), and discordant couples (DC). For the Protocol C samples, we utilized near full-length single genome (NFLG) amplification to generate 288 HIV-1 amplicons from 26 acutely infected seroconverters (SC), while for the 21 recent seroconverter samples (13 from HRW, two from DC, and six from MSM), we PCR amplified overlapping half-genomes. Using PacBio SMRT technology combined with the MDPseq workflow, we performed multiplex sequencing to obtain high accuracy sequences for each amplicon. Phylogenetic analyses indicated that the majority of recent transmitted viruses from DC and HRW clustered within those of the earlier Protocol C cohort. However, five of six sequences from the MSM cohort branched together and were greater than 97% identical. Recombination analyses revealed a high frequency (6/26; 23%) of unique inter-subtype recombination in Protocol C with 19% AC and 4% CD recombinant viruses, which contrasted with only 6.5% of recombinants defined by sequencing of the pol gene previously. The frequency of recombinants was significantly higher (12/21; 57%) in the more recent isolates, although, the five related viruses from the MSM cohort had identical recombination break points. While major drug resistance mutations were absent from Protocol C viruses, 4/21 of recent isolates exhibited transmitted nevirapine resistance. These results demonstrate the ongoing evolution and increased prevalence of recombinant and drug resistant transmitted viruses in Rwanda and highlight the importance of defining NFLG sequences to fully understand the nature of TF viruses and in particular the prevalence of unique recombinant forms (URFs) in transmission cohorts.

Validation of a Triplex Pharmacokinetic Assay for Simultaneous Quantitation of HIV-1 Broadly Neutralizing Antibodies PGT121, PGDM1400, and VRC07-523-LS.

The outcome of the recent Antibody Mediated Prevention (AMP) trials that tested infusion of the broadly neutralizing antibody (bnAb) VRC01 provides proof of concept for blocking infection from sensitive HIV-1 strains. These results also open up the possibility that triple combinations of bnAbs such as PGT121, PGDM1400, as well as long-lasting LS variants such as VRC07-523 LS, have immunoprophylactic potential. PGT121 and PGDM1400 target the HIV-1 V3 and V2 glycan regions of the gp120 envelope protein, respectively, while VRC07-523LS targets the HIV-1 CD4 binding site. These bnAbs demonstrate neutralization potency and complementary breadth of HIV-1 strain coverage. An important clinical trial outcome is the accurate measurement of in vivo concentrations of passively infused bnAbs to determine effective doses for therapy and/or prevention. Standardization and validation of this testing method is a key element for clinical studies as is the ability to simultaneously detect multiple bnAbs in a specific manner. Here we report the development of a sensitive, specific, accurate, and precise multiplexed microsphere-based assay that simultaneously quantifies the respective physiological concentrations of passively infused bnAbs in human serum to ultimately define the threshold needed for protection from HIV-1 infection.

Direct identification of HLA-presented CD8 T cell epitopes from transmitted founder HIV-1 variants.

Cytotoxic T lymphocytes (CTLs) are a critical arm of the immune response to viral infections. The activation and expansion of antigen specific CTL requires recognition of peptide antigens presented on class I major histocompatibility complex molecules (MHC-1) of infected cells. Methods to identify presented peptide antigens that do not rely on the pre-existence of antigen specific CTL are critical to the development of new vaccines. We infected activated CD4+ T cells with two HIV-1 transmitted founder (TF) isolates and used high-resolution mass spectrometry (MS) to identify HIV peptides bound on MHC-1. Using this approach, we identified 14 MHC-1 bound peptides from across the two TF isolates. Assessment of predicted binding thresholds revealed good association of the identified peptides to the shared HLA alleles between the HIV+ donors and the naïve PBMC sample with three peptides identified through peptide sequencing inducing a CD8 T-cell response (p < 0.05). Direct infection of naïve CD4 cells by HIV TF isolates and sequencing of MHC-I presented peptides by HPLC-MS/MS enables identification of novel peptides that may be missed by alternative epitope mapping strategies and can provide valuable insight in to the first peptides presented by an HIV-infected CD4 cell in the first few days post infection.

Selective HLA restriction enables the evaluation and interpretation of immunogenic breadth at comparable levels to that observed with broader HLA distribution.

Existing approaches to identifying predictive T-cell epitopes have traditionally utilized either 2-digit HLA super-families or more commonly utilizing autologous HLA alleles to facilitate the predictions. However, the use of these criteria may not consider the HLA representation within any target population. Here we propose a modification to concept of utilizing autologous HLA whereby subsets of individuals are selected for their specific HLA allele profiles and the representation they provide within a given population. Using this selective approach to HLA selection and the linkages to specific individuals may enable the design of more targeted experimentalstrategies.

Subtype-specific differences in Gag-protease replication capacity of HIV-1 isolates from East and West Africa.

BACKGROUND: The HIV-1 epidemic in sub-Saharan Africa is heterogeneous with diverse unevenly distributed subtypes and regional differences in prevalence. Subtype-specific differences in disease progression rate and transmission efficiency have been reported, but the underlying biological mechanisms have not been fully characterized. Here, we tested the hypothesis that the subtypes prevalent in the East Africa, where adult prevalence rate is higher, have lower viral replication capacity (VRC) than their West African counterparts where adult prevalence rates are lower. RESULTS: Gag-protease sequencing was performed on 213 and 160 antiretroviral-naïve chronically infected participants from West and East Africa respectively and bioinformatic tools were used to infer subtypes and recombination patterns. VRC of patient-derived gag-protease chimeric viruses from West (n = 178) and East (n = 114) Africa were determined using a green fluorescent protein reporter-based cell assay. Subtype and regional differences in VRC and amino acid variants impacting VRC were identified by statistical methods. CRF02_AG (65%, n = 139), other recombinants (14%, n = 30) and pure subtypes (21%, n = 44) were identified in West Africa. Subtypes A1 (64%, n = 103), D (22%, n = 35), or recombinants (14%, n = 22) were identified in East Africa. Viruses from West Africa had significantly higher VRC compared to those from East Africa (p < 0.0001), with subtype-specific differences found among strains within West and East Africa (p < 0.0001). Recombination patterns showed a preference for subtypes D, G or J rather than subtype A in the p6 region of gag, with evidence that subtype-specific differences in this region impact VRC. Furthermore, the Gag A83V polymorphism was associated with reduced VRC in CRF02_AG. HLA-A*23:01 (p = 0.0014) and HLA-C*07:01 (p = 0.002) were associated with lower VRC in subtype A infected individuals from East Africa. CONCLUSIONS: Although prevalent viruses from West Africa displayed higher VRC than those from East Africa consistent with the hypothesis that lower VRC is associated with higher population prevalence, the predominant CRF02_AG strain in West Africa displayed higher VRC than other prevalent strains suggesting that VRC alone does not explain population prevalence. The study identified viral and host genetic determinants of virus replication capacity for HIV-1 CRF02_AG and subtype A respectively, which may have relevance for vaccine strategies.

A Novel Sample Selection Approach to Aid the Identification of Factors That Correlate With the Control of HIV-1 Infection.

Individuals infected with HIV display varying rates of viral control and disease progression, with a small percentage of individuals being able to spontaneously control infection in the absence of treatment. In attempting to define the correlates associated with natural protection against HIV, extreme heterogeneity in the datasets generated from systems methodologies can be further complicated by the inherent variability encountered at the population, individual, cellular and molecular levels. Furthermore, such studies have been limited by the paucity of well-characterised samples and linked epidemiological data, including duration of infection and clinical outcomes. To address this, we selected 10 volunteers who rapidly and persistently controlled HIV, and 10 volunteers each, from two control groups who failed to control (based on set point viral loads) from an acute and early HIV prospective cohort from East and Southern Africa. A propensity score matching approach was applied to control for the influence of five factors (age, risk group, virus subtype, gender, and country) known to influence disease progression on causal observations. Fifty-two plasma proteins were assessed at two timepoints in the 1st year of infection. We independently confirmed factors known to influence disease progression such as the B*57 HLA Class I allele, and infecting virus Subtype. We demonstrated associations between circulating levels of MIP-1α and IL-17C, and the ability to control infection. IL-17C has not been described previously within the context of HIV control, making it an interesting target for future studies to understand HIV infection and transmission. An in-depth systems analysis is now underway to fully characterise host, viral and immunological factors contributing to control.