Exosomes in osteoarthritis: Updated insights on pathogenesis, diagnosis, and treatment.

Osteoarthritis (OA) has remained a prevalent public health problem worldwide over the past decades. OA is a global challenge because its specific pathogenesis is unclear, and no effective disease-modifying drugs are currently available. Exosomes are small and single-membrane vesicles secreted via the formation of endocytic vesicles and multivesicular bodies (MVBs), which are eventually released when MVBs fuse with the plasma membrane. Exosomes contain various integral surface proteins derived from cells, intercellular proteins, DNAs, RNAs, amino acids, and metabolites. By transferring complex constituents and promoting macrophages to generate chemokines and proinflammatory cytokines, exosomes function in pathophysiological processes in OA, including local inflammation, cartilage calcification and degradation of osteoarthritic joints. Exosomes are also detected in synovial fluid and plasma, and their levels continuously change with OA progression. Thus, exosomes, specifically exosomal miRNAs and lncRNAs, potentially represent multicomponent diagnostic biomarkers for OA. Exosomes derived from various types of mesenchymal stem cells and other cell or tissue types affect angiogenesis, inflammation, and bone remodeling. These exosomes exhibit promising capabilities to restore OA cartilage, attenuate inflammation, and balance cartilage matrix formation and degradation, thus demonstrating therapeutic potential in OA. In combination with biocompatible and highly adhesive materials, such as hydrogels and cryogels, exosomes may facilitate cartilage tissue engineering therapies for OA. Based on numerous recent studies, we summarized the latent mechanisms and clinical value of exosomes in OA in this review.

Interleukin-21 enhances the antibody avidity elicited by DNA prime and MVA boost vaccine.

Enhancement of the magnitude or affinity of protective antibodies (Abs) induced by vaccine adjuvant is highly desirable to prevent challenging pathogens such as HIV-1. IL-21 plays a crucial role in germinal center reactions during humoral immune responses. However, the effect of IL-21 as a vaccine adjuvant on the quantity and quality of antigen-specific Abs elicited by DNA prime and MVA boost vaccine, a commonly used vaccine strategy, remains unknown. To close this knowledge gap, female adult B6N mice were primed with DNA vaccine twice (days 0, 14, 100 µg, I.M.) and boosted with MVA vaccine (day 28, 2 × 107 pfu, I.M.) with or without an IL-21 DNA adjuvant (days 3, 17, 31, 40 µg, I.M.), in which HIV-1 gag was expressed as a model antigen. With the addition of an IL-21 adjuvant, we found significantly increased avidity of antigen-specific Abs at multiple time points in a longitudinal follow up. Collectively, our results suggest that an IL-21 immune adjuvant can significantly increase Ab quality induced by heterologous DNA-MVA prime-boost vaccine strategy.

Nanoencapsulation introduces long-acting phenomenon to tenofovir alafenamide and emtricitabine drug combination: A comparative pre-exposure prophylaxis efficacy study against HIV-1 vaginal transmission.

Daily oral antiretroviral (ARV) drugs for pre-exposure prophylaxis (PrEP) has proven efficacy for diverse groups of high-risk individuals. However, daily dosing regimen has augmented non-adherence. These experiments comparatively investigated the long-acting (LA) PrEP potency of subcutaneous (SubQ) administrated tenofovir alafenamide (TAF) and emtricitabine (FTC) loaded nanoparticles (NPs) to solution in humanized (hu) mice. TAF + FTC NPs and TAF + FTC solution (each drug at 200 mg/kg) were administered to hu-CD34-NSG mice (n = 3/time point) for plasma and tissue pharmacokinetic parameter estimation using LC-MS/MS. NP enhanced tissue ARV assimilation compared to plasma. The same dose was administered for PrEP efficacy in HIV-1 challenged hu-BLT mice (n = 5/group). The hu-BLT mice were vaginally challenged with a transmission-founder (T/F) virus at 5 × 105 TCID50 inoculation, on day 4, 7 and 14 post-SubQ treatments (PT) and were compared to infected-untreated-control hu-BLT mice. By 21 days PT, 100% TAF + FTC solution-treated and control-untreated mice were infected. However, TAF + FTC NPs resulted in significant (p = .0002) protection from HIV-1 (day 4: 80%, day 7 and 14: 60%, respectively) compared to control mice. This proof-of-concept study demonstrated detectable TAF/FTC vaginal levels among TAF + FTC NP-treated hu-BLT mice correlating with prolonged PrEP efficacy, thus establishing long-acting TAF + FTC NPs as a potential PrEP modality.

Long-acting parenteral combination antiretroviral loaded nano-drug delivery system to treat chronic HIV-1 infection: A humanized mouse model study.

Human immunodeficiency virus (HIV) patients are often diagnosed in the chronic stage of HIV/AIDS. Combination antiretroviral therapy (cART) has improved quality of life for HIV-infected patients. Present study describes a novel long-acting parenteral formulation of combination antiretroviral (cARV) loaded nano-drugs for treating chronic HIV-1 (cHIV) in a humanized-BLT (hu-BLT) mice model. The cARV (elvitegravir+tenofovir alafenamide+emtricitabine; EVG+TAF+FTC) drugs (mimicking marketed Genvoya® one-pill for HIV-treatment) were encapsulated in poly (lactic-co-glycolic acid) nanoparticles (NPs). To establish cHIV, hu-BLT mice were intravaginally challenged with HIV-1 and maintained for 15 weeks. Plasma viral load (pVL) was monitored by RT-PCR to confirm cHIV. Baseline pVL (week 15) was comparable between treated (n = 10) and control (n = 5) mice groups. Subsequently, treatment hu-BLT mice received 3 subcutaneous doses of cARV NPs (417 mg/kg per dose; n = 10), biweekly, and a fourth/terminal dose a week later. Prior to each treatment and on sacrifice (week 24), pVL was determined. Within three subcutaneous doses of cARV NPs, a non-detectable pVL was established (week 19) and continued until week 22. After the establishment of a non-detectable pVL (week 19-22), 4 treated-mice were sacrificed for tissue drug concentration determination by LC-MS/MS analysis. A considerable amount of cARV was detected at the HIV-infection target and reservoir organs. Subsequently, pVL rebounded comparable to control group by week 24, (7 weeks post-terminal dosage). The present study demonstrated cARV NPs augments sustained ARV efficacy in the cHIV humanized-mouse model. Therefore, cARV NPs could be a novel delivery system to treat cHIV patients, by overcoming drawbacks of conventional cART.

Altered Ratio of T Follicular Helper Cells to T Follicular Regulatory Cells Correlates with Autoreactive Antibody Response in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Individuals with chronic HIV-1 infection have an increased prevalence of autoreactive Abs. Many of the isolated HIV broadly neutralizing Abs from these individuals are also autoreactive. However, the underlying mechanism(s) that produce these autoreactive broadly neutralizing Abs remains largely unknown. The highly regulated coordination among B cells, T follicular helper (TFH) cells, and T follicular regulatory (TFR) cells in germinal centers (GCs) of peripheral lymphatic tissues (LTs) is essential for defense against pathogens while also restricting autoreactive responses. We hypothesized that an altered ratio of TFH/TFR cells in the GC contributes to the increased prevalence of autoreactive Abs in chronic HIV infection. We tested this hypothesis using a rhesus macaque (RM) SIV model. We measured the frequency of TFH cells, TFR cells, and GC B cells in LTs and anti-dsDNA and anti-phospholipid Abs from Indian RMs, with and without SIV infection. We found that the frequency of anti-dsDNA and anti-phospholipid Abs was much higher in chronically infected RMs (83.3% [5/6] and 66.7% [4/6]) than in acutely infected RMs (33.3% [2/6] and 18.6% [1/6]) and uninfected RMs (0% [0/6] and 18.6% [1/6]). The increased ratio of TFH/TFR cells in SIV infection correlated with anti-dsDNA and anti-phospholipid autoreactive Ab levels, whereas the frequency of TFR cells alone did not correlate with the levels of autoreactive Abs. Our results provide direct evidence that the ratio of TFH/TFR cells in LTs is critical for regulating autoreactive Ab production in chronic SIV infection and possibly, by extension, in chronic HIV-1 infection.

SIVcpz closely related to the ancestral HIV-1 is less or non-pathogenic to humans in a hu-BLT mouse model.

The HIV-1 pandemic is a consequence of the cross-species transmission of simian immunodeficiency virus in wild chimpanzees (SIVcpz) to humans. Our previous study demonstrated SIVcpz strains that are closely related to the ancestral viruses of HIV-1 groups M (SIVcpzMB897) and N (SIVcpzEK505) and two SIVcpz lineages that are not associated with any known HIV-1 infections in humans (SIVcpzMT145 and SIVcpzBF1167), all can readily infect and robustly replicate in the humanized-BLT mouse model of humans. However, the comparative pathogenicity of different SIVcpz strains remains unknown. Herein, we compared the pathogenicity of the above four SIVcpz strains with HIV-1 using humanized-BLT mice. Unexpectedly, we found that all four SIVcpz strains were significantly less pathogenic or non-pathogenic compared to HIV-1, manifesting lower degrees of CD4+ T-cell depletion and immune activation. Transcriptome analyses of CD4+ T cells from hu-BLT mice infected with SIVcpz versus HIV-1 revealed enhanced expression of genes related to cell survival and reduced inflammation/immune activation in SIVcpz-infected mice. Together, our study results demonstrate for the first time that SIVcpz is significantly less or non-pathogenic to human immune cells compared to HIV-1. Our findings lay the groundwork for a possible new understanding of the evolutionary origins of HIV-1, where the initial SIVcpz cross-species transmission virus may be initially less pathogenic to humans.

A DNA Vaccine Expressing Consensus Hemagglutinin-Esterase Fusion Protein Protected Guinea Pigs from Infection by Two Lineages of Influenza D Virus.

Two lineages of influenza D virus (IDV) have been found to infect cattle and promote bovine respiratory disease complex, one of the most commonly diagnosed causes of morbidity and mortality within the cattle industry. Furthermore, IDV can infect other economically important domestic livestock, including pigs, and has the potential to infect humans, which necessitates the need for an efficacious vaccine. In this study, we designed a DNA vaccine expressing consensus hemagglutinin-esterase fusion (HEF) protein (FluD-Vax) and tested its protective efficacy against two lineages of IDV (D/OK and D/660) in guinea pigs. Animals that received FluD-Vax (n = 12) developed appreciable titers of neutralizing antibodies against IDV lineage representatives, D/OK and D/660. Importantly, vaccinated animals were protected against intranasal challenge with IDV [3 × 105 50% tissue culture infective dose(s) (TCID50)] D/OK (n = 6) or D/600 (n = 6), based on the absence of viral RNA in necropsied tissues (5 and 7 days postchallenge) using quantitative reverse transcription-PCR and in situ hybridization. In contrast, animals that received a sham DNA vaccine (n = 12) had no detectable neutralizing antibodies against IDV, and viral RNA was readily detectable in respiratory tract tissues after intranasal challenge (3 × 105 TCID50) with IDV D/OK (n = 6) or D/660 (n = 6). Using a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, we found that IDV D/OK and D/600 infections induced apoptosis in epithelial cells lining alveoli and bronchioles, as well as nonepithelial cells in lung tissues. Our results demonstrate for the first time that the consensus IDV HEF DNA vaccine can elicit complete protection against infection from two lineages of IDV in the guinea pig model.IMPORTANCE Influenza D virus (IDV) infection has been associated with bovine respiratory disease complex, one of the most devastating diseases of the cattle population. Moreover, with broad host range and high environmental stability, IDV has the potential to further gain virulence or even infect humans. An efficacious vaccine is needed to prevent infection and stop potential cross-species transmission. In this study, we designed a DNA vaccine encoding the consensus hemagglutinin-esterase fusion (HEF) protein of two lineages of IDV (D/OK and D/660) and tested its efficacy in a guinea pig model. Our results showed that the consensus DNA vaccine elicited high-titer neutralizing antibodies and achieved sterilizing protection against two lineage-representative IDV intranasal infections. To our knowledge, this is the first study showing that a DNA vaccine expressing consensus HEF is efficacious in preventing different lineages of IDV infections.

Associations of Simian Immunodeficiency Virus (SIV)-Specific Follicular CD8+ T Cells with Other Follicular T Cells Suggest Complex Contributions to SIV Viremia Control.

Follicular CD8+ T (fCD8) cells reside within B cell follicles and are thought to be immune-privileged sites of HIV/SIV infection. We have observed comparable levels of fCD8 cells between chronically SIV-infected rhesus macaques with low viral loads (LVL) and high viral loads (HVL), raising the question concerning their contribution to viremia control. In this study, we sought to clarify the role of SIV-specific fCD8 cells in lymph nodes during the course of SIV infection in rhesus macaques. We observed that fCD8 cells, T follicular helper (Tfh) cells, and T follicular regulatory cells (Tfreg) were all elevated in chronic SIV infection. fCD8 cells of LVL animals tended to express more Gag-specific granzyme B and exhibited significantly greater killing than did HVL animals, and their cell frequencies were negatively correlated with viremia, suggesting a role in viremia control. Env- and Gag-specific IL-21+ Tfh of LVL but not HVL macaques negatively correlated with viral load, suggesting better provision of T cell help to fCD8 cells. Tfreg positively correlated with fCD8 cells in LVL animals and negatively correlated with viremia, suggesting a potential benefit of Tfreg via suppression of chronic inflammation. In contrast, in HVL macaques, Tfreg and fCD8 cell frequencies tended to be negatively correlated, and a positive correlation was seen between Tfreg number and viremia, suggesting possible dysfunction and suppression of an effective fCD8 cell immune response. Our data suggest that control of virus-infected cells in B cell follicles not only depends on fCD8 cell cytotoxicity but also on complex fCD8 cell associations with Tfh cells and Tfreg.

Tenofovir alafenamide and elvitegravir loaded nanoparticles for long-acting prevention of HIV-1 vaginal transmission.

OBJECTIVE: This report presents tenofovir (TFV) alafenamide (TAF) and elvitegravir (EVG) fabricated into nanoparticles for subcutaneous delivery as prevention strategy. DESIGN: Prospective prevention study in humanized bone marrow-liver-thymus (hu-BLT) mice. METHODS: Using an oil-in-water emulsion solvent evaporation technique, TAF + EVG drugs were entrapped together into nanoparticles containing poly(lactic-co-glycolic acid). In-vitro prophylaxis studies (90% inhibition concentration) compared nanoparticles with drugs in solution. Hu-BLT (n = 5/group) mice were given 200 mg/kg subcutaneous, and vaginally challenged with HIV-1 [5 × 10 tissue culture infectious dose for 50% of cells cultures (TCID50)] 4 and 14 days post-nanoparticle administration (post-nanoparticle injection). Control mice (n = 5) were challenged at 4 days. Weekly plasma viral load was performed using RT-PCR. Hu-BLT mice were sacrificed and lymph nodes were harvested for HIV-1 viral RNA detection by in-situ hybridization. In parallel, CD34 humanized mice (3/time point) compared TFV and EVG drug levels in vaginal tissues from nanoparticles and solution. TFV and EVG were analyzed from tissue using liquid chromatograph-tandem mass spectrometry (LC-MS/MS). RESULTS: TAF + EVG nanoparticles were less than 200 nm in size. In-vitro prophylaxis indicates TAF + EVG nanoparticles 90% inhibition concentration was 0.002 µg/ml and TAF + EVG solution was 0.78 µg/ml. TAF + EVG nanoparticles demonstrated detectable drugs for 14 days and 72 h for solution, respectively. All hu-BLT control mice became infected within 14 days after HIV-1 challenge. In contrast, hu-BLT mice that received nanoparticles and challenged at 4 days post-nanoparticle injection, 100% were uninfected, and 60% challenged at 14 days post-nanoparticle injection were uninfected (P = 0.007; Mantel-Cox test). In-situ hybridization confirmed these results. CONCLUSION: This proof-of-concept study demonstrated sustained protection for TAF + EVG nanoparticles in a hu-BLT mouse model of HIV vaginal transmission.

Recapitulating Cross-Species Transmission of Simian Immunodeficiency Virus SIVcpz to Humans by Using Humanized BLT Mice.

UNLABELLED: The origins of human immunodeficiency virus type 1 (HIV-1) have been widely accepted to be the consequences of simian immunodeficiency viruses from wild chimpanzees (SIVcpz) crossing over to humans. However, there has not been any in vivo study of SIVcpz infection of humans. Also, it remains largely unknown why only specific SIVcpz strains have achieved cross-species transmission and what transmission risk might exist for those SIVcpz strains that have not been found to infect humans. Closing this knowledge gap is essential for better understanding cross-species transmission and predicting the likelihood of additional cross-species transmissions of SIV into humans. Here we show that humanized bone marrow, thymus, and liver (hu-BLT) mice are susceptible to all studied strains of SIVcpz, including the inferred ancestral viruses of pandemic and nonpandemic HIV-1 groups M (SIVcpzMB897) and N (SIVcpzEK505) as well as strains that have not been found in humans (SIVcpzMT145 and SIVcpzBF1167). Importantly, the ability of SIVcpz to cross the interspecies barrier to infect humanized mice correlates with their phylogenetic distance to pandemic HIV-1. We also identified mutations of SIVcpzMB897 (Env G411R and G413R) and SIVcpzBF1167 (Env H280Q and Q380R) at 14 weeks postinoculation. Together, our results have recapitulated the events of SIVcpz cross-species transmission to humans and identified mutations that occurred during the first 16 weeks of infection, providing in vivo experimental evidence that the origins of HIV-1 are the consequence of SIVcpz crossing over to humans. This study also revealed that SIVcpz viruses whose inferred descendants have not been found in humans still have the potential to cause an HIV-1-like zoonosis. IMPORTANCE: It is believed that the origins of HIV-1 are the consequence of SIV from wild chimpanzees crossing over to humans. However, the origins of HIV-1 have been linked back to only specific SIVcpz strains. There have been no experiments that directly test the in vivo cross-species transmissibility of SIVcpz strains to humans. This is the first in vivo study of SIVcpz cross-species transmission. With the humanized-BLT mouse model, we have provided in vivo experimental evidence of multiple SIVcpz strains crossing over to humans and identified several important mutations of divergent SIVcpz strains after long-term replication in human cells. We also found that the cross-species transmission barrier of SIVcpz to humans correlates with their phylogenetic distance to pandemic HIV-1 group M. Importantly, this work provides evidence that SIVcpz viruses, whose inferred descendants have not been found in humans, still have the potential to cause a future HIV-1-like zoonotic outbreak.

VRC01 antibody protects against vaginal and rectal transmission of human immunodeficiency virus 1 in hu-BLT mice.

Broadly neutralizing antibodies (bNAbs) represent a new generation of antiviral agents for the prevention and treatment of human immunodeficiency virus 1 (HIV-1) infection. A better understanding of the in vivo efficacy of HIV-1 bNAbs, such as VRC01, in preventing mucosal transmission of HIV-1 has important implications for HIV-1 vaccine design. In this study, we evaluated the efficacy of passively transferred VRC01 antibody in preventing HIV-1 vaginal and rectal transmission in humanized bone marrow/liver/thymus mice (hu-BLT mice). Mice were subcutaneously injected with VRC01 IgG, and 24 hours later, they were challenged intravaginally or intrarectally with HIV-1Ada. All hu-BLT mice receiving VRC01 IgG antibody were aviremic at 2 weeks after intravaginal (n = 3) or intrarectal (n = 6) challenge as measured by quantitative real-time RT-PCR. In contrast, mice receiving control IgG all became infected. By 5 and 6 weeks post-challenge, some of VRC01 aviremic mice in both the intravaginal and intrarectal challenge groups became viremic. Our results suggest that VRC01 antibody can be protective against HIV-1 vaginal and rectal transmission; however, a single administration of VRC01 cannot completely prevent mucosal infection.

Early Initiation of Antiretroviral Therapy Can Functionally Control Productive HIV-1 Infection in Humanized-BLT Mice.

BACKGROUND: Recent reports showed that functional control of HIV-1 infection for a prolonged time is possible by early antiretroviral therapy (ART); however, its underlying mechanism needs to be studied with a suitable animal model. Recently, humanized-BLT (bone marrow, liver, and thymus) mouse (hu-BLT) was shown to be an excellent model for studying HIV-1 infection. We thus tested the feasibility of studying functional control of HIV-1 infection using hu-BLT mice. METHODS: Animals in 3 treatment groups (Rx-6h, Rx-24h, and Rx-48h) and untreated group were infected with HIV-1, followed by ART initiation at 6, 24, or 48 hours postinfection and continued daily for 2 weeks. Three weeks after stopping ART, CD8 T cells were depleted from all animals. Plasma viral load was monitored weekly using droplet digital polymerase chain reaction. Percentage of CD4 and CD8 T cells were measured by flow cytometry. In situ hybridization and droplet digital polymerase chain reaction were used to detect viral RNA (vRNA) and DNA. RESULTS: Although control animals had high viremia throughout the study, all Rx-6h animals had undetectable plasma viral load after ART cessation. After CD8 T-cell depletion, viremia increased and CD4 T cells decreased in all animals except the Rx-6h group. Viral DNA was detected in spleens of all animals and a few vRNA cells were detected by in situ hybridization in 1 of 3 Rx-6h animals. CONCLUSIONS: Early ART did not act as prophylaxes but, rather, can control HIV-1 productive infection and prevented CD4 T-cell depletion in hu-BLT mice. This mouse model can be used to elucidate the mechanism for functional control of HIV-1.