Background: The lungs were historically identified as one of the major anatomic sites for HIV replication in the pre-antiretroviral therapy (ART) era. However, their contribution to HIV persistence in individuals under suppressive ART remains understudied. Design: We assessed HIV persistence and comprehensively characterized pulmonary mucosal CD4⁺ T cells in HIV-infected (HIV⁺) individuals receiving long-term suppressive ART versus uninfected participants. Methods: Bronchoalveolar lavage (BAL), bronchial biopsies, and matched peripheral blood were obtained from n = 24 HIV-infected adults receiving long-term suppressive ART (median: 9 years) and n = 8 healthy volunteers without respiratory symptoms. HIV-DNA and cell-associated HIV-RNA were quantified by ultra-sensitive PCR, and lung mucosal CD4⁺ T-cell subsets were characterized by multiparameter flow cytometry. Results: The levels of HIV-DNA were 13-fold higher in total BAL cells compared to blood. Importantly, FACS-sorted CD4⁺ T cells from BAL contained greater levels of HIV-DNA compared to peripheral CD4⁺ T cells. BAL CD4⁺ T cells in HIV⁺ individuals were characterized mostly by an effector memory phenotype, whereas naive and terminally differentiated cells were underrepresented compared to blood. Furthermore, BAL CD4⁺ T cells expressed higher levels of immune activation (HLA-DR/CD38) and senescence (CD57) markers. Importantly, BAL was enriched in T-cell subsets proposed to be preferential cellular HIV reservoirs, including memory CD4⁺CCR6⁺, Th1Th17 (CD4⁺CCR6⁺CCR4⁻CXCR3⁺), CD4⁺CCR6⁺CXCR3⁻CCR4⁻, and CD4⁺CD32a⁺ T cells. Conclusion: The pulmonary mucosa represents an important immunological effector site highly enriched in activated and preferential CD4⁺ T-cell subsets for HIV persistence during long-term ART in individuals without respiratory symptoms. Our findings raise new challenges for the design of novel HIV eradication strategies in mucosal tissues.

Increased frequencies of immunosuppressive regulatory T cells (Tregs) are associated with gut lymphoid tissue fibrosis and dysfunction which, in turn, contribute to disease progression in chronic simian immunodeficiency virus/human immunodeficiency virus (SIV/HIV) infection. Mesenteric lymph nodes (MLNs), which drain the large and small intestine, are critical sites for the induction and maintenance of gut mucosal immunity. However, the dynamics of Tregs in MLNs are not well understood due to the lack of accessibility to these tissues in HIV-infected individuals. Here, the dynamics of Tregs in blood and MLNs were assessed in SIV-infected rhesus macaques (RMs) following early antiretroviral drug (ARV) initiation. Early ARV initiation reduced T-cell immune activation, as assessed by HLA-DR/CD39 expression, and prevented the depletion of memory CCR6⁺ Th17 cells in both blood and MLNs. Untreated animals showed higher frequencies of Tregs, CD39⁺ Tregs, thymic Tregs, and new memory CD4 populations sharing similarity with Tregs as CTLA4⁺ PD1^– and CTLA4⁺ PD1^– FoxP3⁺ T cells. Despite early ARV treatment, the frequencies of these Treg subsets remained unchanged within the MLNs and, in contrast to blood normalization, the Th17/Treg ratio remained distorted in MLNs. Furthermore, our results highlighted that the expressions of IDO-1, TGFβ1 and collagen-1 mRNA remained unchanged in MLN of ARV-treated RMs. ARV interruption did not affect T-cell immune activation and Th17/Treg ratios in MLN. Altogether, our data demonstrated that early ARV initiation within the first few days of SIV infection is unable to reduce the frequencies and homing of various subsets of Tregs within the MLNs which, in turn, may result in tissue fibrosis, impairment in MLN function, and HIV persistence. IMPORTANCE Tregs contribute to SIV/HIV disease progression by inhibition of antiviral specific responses and effector T-cell proliferation. Tregs also cause tissue fibrosis via transforming growth factor β1 production and collagen deposition, which are associated with microbial translocation and generalized immune activation. Early ARV initiation upon viral exposure is recommended globally and results in improved immune function recovery and reduced viral persistence. Here, using an acute SIV infection model of rhesus macaques, we demonstrated for the first time that despite clear improvements in mucosal CD4 T cells, in contrast to blood, Treg frequencies in MLNs remained elevated following early ARV initiation. The particular Th17/Treg balance observed in MLNs can contribute, in part, to the maintenance of mucosal fibrosis during suppressive ARV treatment. Our results provide a better understanding of gut mucosal immune dynamics following early ARV initiation. These findings suggest that Treg-based treatments could serve as a novel immunotherapeutic approach to decrease gut mucosal damage during SIV/HIV infections.

Background: The cattle tick, Rhipicephalus (Boophilus) microplus, affects livestock production in many regions of the world. Up to now, the widespread use of chemical acaricides has led to the selection of acaricide-resistant ticks and to environmental contamination. Gavac^plus is a subunit vaccine based on the recombinant Bm86 tick antigen expressed in yeast, capable to control infestations of R. microplus under controlled and production conditions. The vaccine constitutes the core element of broad control programs against this ectoparasite, in which acquired immunity in cattle to Bm86 is combined with a rational use of acaricides. At present, the conventional vaccine scheme consists of three doses that should be administered at weeks 0, 4 and 7, followed by a booster every six months. Results: In this study we assayed a reduction in the number of the initial doses of Gavac^plus, evaluated the time course and the level of bovine anti-Bm86 antibodies elicited, and analyzed the vaccine effect on ticks engorging on immunized cattle under production conditions. Following three different immunization schemes, the bovines developed a strong and specific immune response characterized by elevated anti-Bm86 IgG titers. A reduction in the weight of engorging female ticks, in the weight of the eggs laid and also in R. microplus viable eggs percentage was obtained by using only two doses of Gavac^plus administered at weeks 0 and 4, followed by a booster six months later. This reduction did not differ from the results obtained on ticks engorging on cattle immunized at weeks 0, 4 and 7. It was also demonstrated that anti-Bm86 antibody titers over 1:640, measured in bovines immunized at weeks 0 and 4, were sufficient to affect weight and reproductive potential of female ticks as compared with ticks engorging on unvaccinated animals. In addition, no statistically significant differences were detected in the average weight of eggs laid by ticks engorged on immunized cattle that showed anti-Bm86 specific titers in the range of 1:640 to 1:81920. Conclusion: The administration of two initial doses of Gavac^plus containing 100 μg of Bm86 antigen to non-immunized cattle under production conditions is sufficient to affect the weight and the reproductive capacity of R. microplus engorging females. According to these results, cattle herds' manipulation and vaccine costs could be potentially reduced with a positive impact on the implementation of integrated control programs against R. microplus.

Vaccine design strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are focused on the Spike protein or its subunits as the main antigen target of neutralizing antibodies. In this work, we propose rapid production methods of an extended segment of the Spike Receptor Binding Domain (RBD) in HEK293SF cells cultured in suspension, in serum-free media, as a major component of a COVID-19 subunit vaccine under development. The expression of RBD, engineered with a sortase-recognition motif for protein-based carrier coupling, was achieved at high yields by plasmid transient transfection or human type-5-adenoviral infection of the cells, in a period of only two and three weeks, respectively. Both production methods were evaluated in 3L-controlled bioreactors with upstream and downstream bioprocess improvements, resulting in a product recovery with over 95% purity. Adenoviral infection led to over 100 µg/mL of RBD in culture supernatants, which was around 7-fold higher than levels obtained in transfected cultures. The monosaccharide and sialic acid content was similar in the RBD protein from the two production approaches. It also exhibited a proper conformational structure as recognized by monoclonal antibodies directed against key native Spike epitopes. Efficient direct binding to ACE2 was also demonstrated at similar levels in RBD obtained from both methods and from different production lots. Overall, we provide bioprocess-related data for the rapid, scalable manufacturing of low cost RBD based vaccines against SARS-CoV-2, with the added value of making a functional antigen available to support further research on uncovering mechanisms of virus binding and entry as well as screening for potential COVID-19 therapeutics.