Search

We previously developed an innovative strategy to induce CD8 + T lymphocyte-immunity through in vivo engineering of extracellular vesicles (EVs). This approach relies on intramuscular injection of DNA expressing antigens of interest fused at a biologically-inactive HIV-1 Nef protein mutant (Nef mut ). Nef mut is very efficiently incorporated into EVs, thus conveying large amounts of fusion proteins into EVs released by transfected cells. This platform proved successful against highly immunogenic tumor-specific antigens. Here, we tested whether antigen-specific CD8 + T cell immune responses induced by engineered EVs can counteract the growth of tumors expressing two "self" tumor-associated antigens (TAAs): HOXB7 and Her2/neu. FVB/N mice were injected with DNA vectors expressing Nef mut fused to HOXB7 or Her2/neu, singly and in combination, before subcutaneous implantation of breast carcinoma cells co-expressing HOXB7 and Her2/neu. All mice immunized with the combination vaccine remained tumor-free, whereas groups vaccinated with single Nef mut -fused antigens were only partly protected, with stronger antitumor effects in Her2/neu-immunized mice. Double-vaccinated mice also controlled tumor growth upon a later tumor cell re-challenge. Importantly, co-vaccination also contained tumors in a therapeutic immunization setting. These results showed the efficacy of EV-based vaccination against two TAAs, and represent the first demonstration that HOXB7 may be targeted in multi-antigen immunotherapy strategies., Competing Interests: Competing interests The authors declare no competing interests. Ethics approval and consent to participate The authors declare that all methods were performed in accordance with the relevant guidelines and regulations and that the study on animals was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Italian Ministry of Health, authorization 78/2020, released on February 11th, 2020., (© 2024. The Author(s).)

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 enters the host by infecting nasal ciliated cells. Then, the virus can spread towards the oropharyngeal cavity and the pulmonary tissues. The antiviral adaptive immunity is promptly induced in response to the virus's detection, with virus-specific T-lymphocytes appearing before antiviral antibodies. Both the breadth and potency of antiviral CD8 + T-cell immunity have a key role in containing viral spread and disease severity. Current anti-SARS-CoV-2 vaccines do not impede the virus's replication in the upper respiratory tract, and there is consensus on the fact that the best potency of the antiviral immune response in both blood and the upper respiratory tract can be reached upon infection in vaccinees (i.e., breakthrough infection). However, whether the antiviral CD8 + T-cells developing in response to the breakthrough infection in the upper respiratory tract diffuse to the lungs is also still largely unknown. To fill the gap, we checked the CD8 + T-cell immunity elicited after infection of K18-hACE2 transgenic mice both at 3 weeks and 3 months after anti-spike vaccination. Virus-specific CD8 + T-cell immunity was monitored in both blood and the lungs before and after infection. By investigating the de novo generation of the CD8 + T-cells specific for SARS-CoV-2 viral proteins, we found that both membrane (M) and/or nucleocapsid (N)-specific CD8 + T-cells were induced at comparable levels in the blood of both unvaccinated and vaccinated mice. Conversely, N-specific CD8 + T-cells were readily found in the lungs of the control mice but were either rare or absent in those of vaccinated mice. These results support the idea that the hybrid cell immunity developing after asymptomatic/mild breakthrough infection strengthens the antiviral cell immunity in the lungs only marginally, implying that the direct exposition of viral antigens is required for the induction of an efficient antiviral cell immunity in the lungs.

Induction of effective immunity in the lungs should be a requisite for any vaccine designed to control the severe pathogenic effects generated by respiratory infectious agents. We recently provided evidence that the generation of endogenous extracellular vesicles (EVs) engineered for the incorporation of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 Nucleocapsid (N) protein induced immunity in the lungs of K18-hACE2 transgenic mice, which then can survive the lethal virus infection. However, nothing is known about the ability of the N-specific CD8 + T cell immunity in controlling viral replication in the lungs, a major pathogenic signature of severe disease in humans. To fill the gap, we investigated the immunity generated in the lungs by N-engineered EVs in terms of induction of N-specific effectors and resident memory CD8 + T lymphocytes before and after virus challenge carried out three weeks and three months after boosting. At the same time points, viral replication extents in the lungs were evaluated. Three weeks after the second immunization, virus replication was reduced in mice best responding to vaccination by more than 3-logs compared to the control group. The impaired viral replication matched with a reduced induction of Spike-specific CD8 + T lymphocytes. The antiviral effect appeared similarly strong when the viral challenge was carried out 3 months after boosting, and associated with the persistence of N-specific CD8 + T-resident memory lymphocytes. In view of the quite low mutation rate of the N protein, the present vaccine strategy has the potential to control the replication of all emerging variants., (© 2023. The Author(s).)

Introduction: Ethiopia has experienced rapid expansion of antiretroviral therapy (ART). However, as long-term retention in ART therapy is key for ART effectiveness, determinants of attrition need to be identified so appropriate interventions can be designed., Methods: We used data from the 'Cohort of African people Starting Antiretroviral therapy' (CASA) project, a prospective study of a cohort of HIV-infected patients who started ART in seven health facilities (HFs). We analysed the data of patients who had started first-line ART between January 2013 and December 2014. The Kaplan-Meier method was used to estimate the probability of retention at different time points. The Cox proportional hazards model was used to identify factors associated with attrition., Results: A total of 1198 patients were included in the study. Kaplan-Meier estimates of retention in care were 83.9%, 82.1% and 79.8% at 12, 18 and 24 months after starting ART, respectively. Attrition was mainly due to loss to follow-up, transferred-out patients and documented mortality. A multivariate Cox proportional hazard model showed that male sex, CD4 count <200 cells/µL and the type of HF were significantly associated with attrition., Conclusions: The observed attrition differences according to gender suggest that separate interventions designed for women and men should be explored. Moreover, innovative strategies to increase HIV testing should be supported to avoid CD4 levels falling too low, a factor significantly associated with higher attrition in our study. Finally, specific studies to analyse the reasons for different levels of attrition among HFs are required., Competing Interests: Competing interests: None declared.

Background: To describe the development and the psychometric properties of the Istituto Superiore di Sanità-HIV symptoms scale (lSS-HIV symptoms scale)., Methods: The ISS-HIV symptom scale was developed by an Italian working team including researchers, physicians and people living with HIV. The development process went through the following steps: (1) review of HIV/AIDS literature; (2) focus group; (3) pre-test analysis; (4) scale validation., Results: The 22 symptoms of HIV-ISS symptoms scale were clustered in five factors: pain/general discomfort (7 items); depression/anxiety (4 items); emotional reaction/psychological distress (5 items); gastrointestinal discomfort (4 items); sexual discomfort (2 items). The internal consistence reliability was for all factors within the minimum accepted standard of 0.70., Conclusions: The results of this study provide a preliminary evidence of the reliability and validity of the ISS-HIV symptoms scale. In the new era where HIV infection has been transformed into a chronic diseases and patients are experiencing a complex range of symptoms, the ISS-HIV symptoms scale may represent an useful tool for a comprehensive symptom assessment with the advantage of being easy to fill out by patients and potentially attractive to physicians mainly because it is easy to understand and requires short time to interpret the results.

Introduction: Although Ethiopia has been scaling up the antiretroviral therapy (ART) services, low retention in care of patients remains one of the main obstacles to treatment success. We report data on retention in care and its associated determinants in Tigray, Ethiopia., Methods: We used data from the CASA project, a prospective observational and multi-site study of a cohort of HIV-infected patients who initiated ART for the first time in Tigray. Four participating health facilities (HFs) located in the South of Tigray were considered for this study. Patients were followed for one year after ART initiation. The main outcome measure was represented by the current retention in care, defined as the proportion of patients who were alive and receiving ART at the same HF one year after ART initiation. Patients who started ART between January 1, 2013 and December 31, 2013 were included in this analysis. Patients were followed for one year after ART initiation. The determinants of retention were analysed using univariate and multivariate Cox Proportional Hazards model with robust sandwich estimates to account for within HF correlation., Results: The four participating HFs in Tigray were able to retain overall 85.1% of their patients after one year from starting ART. Loss to follow-up (5.5%) and transfers to other HF (6.6) were the main determinant of attrition. A multivariate analysis shows that the factors significantly associated with retention were the type of HF, gender and active TB. Alamata health center was the HF with the highest attrition rate (HR 2.99, 95% CI: 2.77-3.23). Active TB (HR 1.72, 95% CI: 1.23-2.41) and gender (HR 1.64, 95% CI: 1.10-2.56) were also significantly associated with attrition., Conclusions: Although Ethiopia has significantly improved access to the ART program, achieving and maintaining a satisfactory long-term retention rate is a future goal. This is difficult because of different retention rates among HFs. Moreover specific interventions should be directed to people of different sex to improve retention in care in male population.

Background: Health-related quality of life (HRQoL) has been recognized as a central measure of the overall health status in HIV patients. With the availability of different highly effective drug combinations, maximizing quality-adjusted survival has become a major target of HIV treatment. Although the association of HIV RNA and CD4 cell count with clinical HIV progression has been well established, the relation between these markers and HRQoL measures is still unclear., Method: This cross-sectional study investigated the relationship linking HIV RNA and CD4 to HRQoL measures in 181 triple-class-experienced patients with advanced HIV disease. The instrument used was the ISSQoL, a self-administered and HIV-specific HRQoL questionnaire., Results: Data showed no correlation between HRQoL measures and CD4 counts. Higher HIV RNA levels were, however, associated with poor HRQoL scores in 3 out of 9 scales of social functioning, depression and anxiety, and satisfaction with quality of life. In multivariable analyses, only the satisfaction with quality of life mean score remained significantly lower for the HIV RNA ≯100,000 copies/mL group compared to the HIV RNA 50 to 10,000 copies/mL group., Conclusions: Although other determinants of HRQoL in people with HIV should also be considered, this finding suggests a negative impact of high viral load on perceived HRQoL that adds to other described determinants of lower quality of life in people with HIV, such as lower social support and self-reported symptoms.

We recently showed that both replicating and resting cells cultivated with ganciclovir (GCV) were killed when challenged with vesicular stomatitis virus G glycoprotein pseudotyped HIV-1-based virus-like particles (VLPs) carrying the Nef7 (i.e., an HIV-1 Nef mutant incorporating in virions at high levels)/herpes simplex virus-1 thymidine kinase (HSV-TK) fusion product. On this basis, a novel anti-HIV therapeutic approach based on Nef7/TK VLPs expressing X4 or R5 HIV cell receptor complexes has been attempted. We here report that (CD4-CXCR4) and (CD4-CCR5) Nef7-based VLPs efficiently enter cells infected by X4- or R5-tropic HIV-1 strains, respectively. Importantly, the delivery of the VLP-associated Nef7/TK led to cell death upon GCV treatment. Of interest, VLPs were effective also against non-replicating, HIV-1-infected primary human monocyte-derived macrophages. HIV-targeted VLPs represent a promising candidate for the treatment of persistently HIV-1-infected cells that are part of virus reservoirs resistant to HAART therapies.

HIV-1 Nef incorporates into virions at low levels, likely about 10 molecules per viral particle. Here, we describe a Nef mutant (Nef7) apparently showing more than 100-fold higher efficiency of virion incorporation. Interestingly, Nef7 can act as a cargo molecule for protein delivery into the cells, as its virion incorporation appeared conserved even upon C-terminal fusion with proteins of up to 30 kDa. This was demonstrated first by assessing the intracellular fluorescence of cells challenged with lentivirus-based virus-like particles (VLPs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) and incorporating Nef7 fused with the green fluorescent protein. Furthermore, the biologic activity of products delivered by Nef7-based VLPs was demonstrated by tagging Nef7 with the herpes simplex virus-1 thymidine kinase (HSV-1 TK). In fact, we observed that both cell lines and primary human macrophages challenged with (VSV-G) Nef7/TK VLPs died after 5 to 7 days of treatment with ganciclovir (GCV). In sum, our findings support the notion that Nef7-based VLPs can be considered platforms for original systems of protein delivery. In particular, the here- described Nef7/TK VLPs represent a first applicative example opening the way toward new HSV-1 TK/GCV-based cell suicide therapies circumventing cell gene engineering procedures.

The expression of human immunodeficiency virus Nef increases the viral infectivity through mechanisms still not fully elucidated. Here we report that wild-type (wt) human immunodeficiency virus, type 1 (HIV-1), particles were neutralized by higher concentrations of either anti-Env glycoprotein (gp) 41 antibodies or recombinant soluble human CD4 compared with Deltanef HIV-1. This appeared to be the result of a Nef-induced increase of virion incorporation of both gp41 (transmembrane (TM)) and surface gp120 Env products likely originating from enhanced steady-state levels of cell membrane-associated Env products. This, in turn, seemed to be the consequence of a reduced retention of the Env precursor. Most interesting, we found that both the Nef-directed increase of Env membrane expression and the Nef-induced enhancement of HIV-1 infectivity relied on the presence of the intracytoplasmic domain of TM, supporting the hypothesis of a functional correlation between these effects. Mutagenesis studies allowed us to establish that the two leucine residues at the TM C terminus, which are part of a sorting motif involved in the control of Env membrane expression, and the 181-210-residue Nef C-terminal region were critically involved in the Nef/Env functional interaction. In conclusion, we propose that Nef increases the infectivity of HIV-1 at least in part by enhancing the amounts of Env products incorporated into virus particles.

Expression of the human immunodeficiency virus type 1 (HIV-1) Nef triple mutant F12Nef strongly inhibits HIV-1 replication. We exploited such a unique feature in a novel anti-HIV-1 gene therapy design by constructing an HIV-1 Tat-defective lentivirus vector expressing the product of fusion between the low-affinity human nerve growth factor receptor truncated in its intracytoplasmic domain (deltaNGFr, NH(2) moiety), and F12Nef (COOH moiety), under the control of the HIV-1 long terminal repeats. In this manner, both the selection marker (deltaNGFr) and the anti-HIV-1 effector are comprised in the same fusion protein, the expression of which is targetable by HIV-1 infection. Such a vector was proved to transduce human cells efficiently and, on HIV-1 infection, it expressed high levels of the fusion protein. In addition, strong antiviral activity of the deltaNGFr/F12Nef-expressing vector was demonstrated in cell lines as well as in primary cell cultures challenged with T- or M-tropic HIV-1 isolates. Thus, the HIV-1-targetable expression of the deltaNGFr/F12Nef fusion protein represents a novel and powerful tool for an effective anti-HIV-1 gene therapy strategy.

Increasing interest has been devoted to the role that monocyte-macrophages play in the pathogenesis of AIDS. The hypothesis of an involvement in AIDS pathogenesis of human/simian immunodeficiency virus (HIV/SIV) Nef also is currently under evaluation by many investigators. The original basis of this hypothesis came from evidence that monkeys infected with a nef-deleted SIV strain failed to develop simian AIDS. Here, we show that treatment of human monocyte-derived macrophages (MDM) with recombinant HIV-1 Nef protein (rNef) induces a strong inhibition of the replication of either macrophage (M-) or dual-tropic HIV-1 strains. Through cytofluorimetric analyses, we detected internalization of FITC-conjugated rNef in MDM as early as 6 h after treatment. Confocal microscope observations demonstrated that the intracellular distribution of internalized rNef was identical to that of endogenously produced Nef. Down-regulation of the CD4 HIV receptor detected upon rNef treatment of MDM suggested that the rNef-induced HIV inhibition occurred at the virus entry step. This deduction was strengthened by the observation that CD4-independent infection was totally insensitive to rNef treatment. The specificity of all observed effects was demonstrated by immunodepletion of rNef. Finally, we showed that the resistance to HIV replication induced by rNef treatment in MDM favours the spread of T-tropic over M-tropic HIV strains in doubly infected CD4(+) lymphocyte-MDM co-cultures. We propose that extracellular Nef contributes to AIDS pathogenesis by inducing resistance to M-tropic HIV replication in MDM, thereby facilitating the switching from M- to T-tropic HIV prevalence that correlates frequently with AIDS progression.