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Previous studies on highly HIV-1-exposed, yet persistently seronegative women from the Punwami Sex Worker cohort in Kenya, have shed light on putative protective mechanisms, suggesting that mucosal immunological factors, such as antiproteases, could be mediating resistance to HIV-1 transmission in the female reproductive tract. Nine protease inhibitors were selected for this study: serpin B4, serpin A1, serpin A3, serpin C1, cystatin A, cystatin B, serpin B13, serpin B1 and α-2-macroglobulin-like-protein 1. We assessed in a pilot study, the activity of these antiproteases with cellular assays and an ex vivo HIV-1 challenge model of human ecto-cervical tissue explants. Preliminary findings with both models, cellular and tissue explants, established an order of inhibitory potency for the mucosal proteins as candidates for pre-exposure prophylaxis when mimicking pre-coital use. Combination of all antiproteases considered in this study was more active than any of the individual mucosal proteins. Furthermore, the migration of cells out of ecto-cervical explants was blocked indicating potential prevention of viral dissemination following amplification of the founder population. These findings constitute the base for further development of these mucosal protease inhibitors for prevention strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2022 Herrera, Olejniczak, Noël-Romas, Plummer and Burgener.)

Natural Killer (NK) cells play a key role in controlling HIV replication, with potential downstream impact on the size of the HIV reservoir and likelihood of viral rebound after antiretroviral therapy (ART) cessation. It is therefore important to understand how primary HIV infection (PHI) disrupts NK cell function, and how these functions are restored by early ART. We examined the impact of commencing ART during PHI on phenotypic and functional NK cell markers at treatment initiation (baseline), 3 months, 1 year, and 2 years in seven well-characterised participants in comparison to HIV seronegative volunteers. We then examined how those NK cell properties differentially impacted by ART related to time to viral rebound and HIV DNA levels in 44 individuals from the SPARTAC trial who stopped ART after 48 weeks treatment, started during PHI. NK cell markers that were significantly different between the seven people with HIV (PWH) treated for 2 years and HIV uninfected individuals included NKG2C levels in CD56 dim NK cells, Tim-3 expression in CD56 bright NK cells, IFN-γ expressed by CD56 dim NK cells after IL-12/IL-18 stimulation and the fraction of Eomes-/T-bet+ in CD56 dim and CD56 bright NK cells. When exploring time to viral rebound after stopping ART among the 44 SPARTAC participants, no single NK phenotypic marker correlated with control. Higher levels of IL-12/IL-18 mediated NK cell degranulation at baseline were associated with longer times to viral rebound after treatment interruption (P=0.028). Additionally, we found higher fractions of CD56 dim NK cells in individuals with lower levels of HIV DNA (P=0.048). NKG2A and NKp30 levels in CD56 neg NK cells were higher in patients with lower HIV DNA levels (p=0.00174, r=-0.49 and p=0.03, r= -0.327, respectively) while CD27 levels were higher in those with higher levels of HIV DNA (p=0.026). These data show NK cell functions are heterogeneously impacted by HIV infection with a mixed picture of resolution on ART, and that while NK cells may affect HIV DNA levels and time to viral rebound, no single NK cell marker defined delayed viral rebound., Competing Interests: Author JH was employed by Etcembly Ltd, Harwell Campus, United Kingdom. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pace, Ogbe, Hurst, Robinson, Meyerowitz, Olejniczak, Thornhill, Jones, Waters, Lwanga, Kuldanek, Hall, Zacharopoulou, Martin, Brown, Nwokolo, Peppa, Fox, Fidler and Frater.)

Non-human primates (NHP) are widely used for the pre-clinical assessment of antiretrovirals (ARVs) for HIV treatment and prevention. However, the utility of these models is questionable given the differences in ARV pharmacology between humans and macaques. Here, we report a model based on ex vivo ARV exposure and the challenge of mucosal tissue explants to define pharmacological differences between NHPs and humans. For colorectal and cervicovaginal explants in both species, high concentrations of tenofovir (TFV) and maraviroc were predictive of anti-viral efficacy. However, their combinations resulted in increased inhibitory potency in NHP when compared to human explants. In NHPs, higher TFV concentrations were measured in colorectal versus cervicovaginal explants (p = 0.042). In humans, this relationship was inverted with lower levels in colorectal tissue (p = 0.027). TFV-resistance caused greater loss of viral fitness for HIV-1 than SIV. This, tissue explants provide an important bridge to refine and appropriately interpret NHP studies., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

T cell dysfunction occurs early following HIV infection, impacting the emergence of non-AIDS morbidities and limiting curative efforts. ART initiated during primary HIV infection (PHI) can reverse this dysfunction, but the extent of recovery is unknown. We studied 66 HIV-infected individuals treated from early PHI with up to three years of ART. Compared with HIV-uninfected controls, CD4 and CD8 T cells from early HIV infection were characterised by T cell activation and increased expression of the immune checkpoint receptors (ICRs) PD1, Tim-3 and TIGIT. Three years of ART lead to partial - but not complete - normalisation of ICR expression, the dynamics of which varied for individual ICRs. For HIV-specific cells, epigenetic profiling of tetramer-sorted CD8 T cells revealed that epigenetic features of exhaustion typically seen in chronic HIV infection were already present early in PHI, and that ART initiation during PHI resulted in only a partial shift of the epigenome to one with more favourable memory characteristics. These findings suggest that although ART initiation during PHI results in significant immune reconstitution, there may be only partial resolution of HIV-related phenotypic and epigenetic changes., Competing Interests: WH is an employee of Merck and Company and holds equity in Tango Therapeutics and Arsenal Biosciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Martin, Sen, Pace, Robinson, Meyerowitz, Adland, Thornhill, Jones, Ogbe, Parolini, Olejniczak, Zacharopoulou, Brown, Willberg, Nwokolo, Fox, Fidler, Haining and Frater.)

Initiation of antiretroviral therapy (ART) in early compared with chronic human immunodeficiency virus (HIV) infection is associated with a smaller HIV reservoir. This longitudinal analysis of 60 individuals who began ART during primary HIV infection (PHI) investigates which pre- and posttherapy factors best predict HIV DNA levels (a correlate of reservoir size) after treatment initiation during PHI. The best predictor of HIV DNA at 1 year was pre-ART HIV DNA, which was in turn significantly associated with CD8 memory T-cell differentiation (effector memory, naive, and T-bet-Eomes- subsets), CD8 T-cell activation (CD38 expression) and T-cell immunoglobulin and mucin-domain containing-3 (Tim-3) expression on memory T cells. No associations were found for any immunological variables after 1 year of ART. Levels of HIV DNA are determined around the time of ART initiation in individuals treated during PHI. CD8 T-cell activation and memory expansion are linked to HIV DNA levels, suggesting the importance of the initial host-viral interplay in eventual reservoir size., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America.)

Gut-associated lymphoid tissue (GALT) is a key location for the HIV reservoir. The observation that B-cell-T-cell doublets are enriched for CD32a (a low-affinity IgG receptor) in peripheral blood raises interesting questions, especially as these cells have been associated with HIV DNA in some studies. We sought to determine if similar doublets were present in GALT, the significance of these doublets, and their implications for the HIV reservoir. Given the importance of GALT as a reservoir for HIV, we looked for expression of CD32 on gut CD4 T cells and for evidence of doublets, and any relationship with HIV DNA in HIV + individuals initiated on antiretroviral therapy (ART) during primary HIV infection (PHI). Tonsil tissue was also available for one individual. As previously shown for blood, CD32 high CD4 cells were mainly doublets of CD4 T cells and B cells, with T-cell expression of ICOS in tonsil and gut tissue. CD4 T cells associated with CD32 (compared with 'CD32-' CD4 cells) had higher expression of follicular markers CXCR5, PD-1, ICOS, and Bcl-6 consistent with a T follicular helper (TFH) phenotype. There was a significant correlation between rectal HIV DNA levels and CD32 expression on TFH cells. Together, these data suggest that CD32 high doublets are primarily composed of TFH cells, a subset known to be preferentially infected by HIV.

Efforts to both characterize and eradicate the HIV reservoir have been limited by the rarity of latently infected cells and the absence of a specific denoting biomarker. CD32a (FcγRIIa) has been proposed to be a marker for an enriched CD4 T cell HIV reservoir, but this finding remains controversial. Here, we explore the expression of CD32 on CD3 + CD4 + cells in participants from two primary HIV infection studies and identify at least three distinct phenotypes (CD32 low , CD32 + CD14 + , and CD32 high ). Of note, CD4 negative enrichment kits remove the majority of CD4 + CD32 + T cells, potentially skewing subsequent analyses if used. CD32 high CD4 T cells had higher levels of HLA-DR and HIV co-receptor expression than other subsets, compatible with their being more susceptible to infection. Surprisingly, they also expressed high levels of CD20, TCRαβ, IgD, and IgM (but not IgG), markers for both T cells and naïve B cells. Compared with other populations, CD32 low cells had a more differentiated memory phenotype and high levels of immune checkpoint receptors, programmed death receptor-1 (PD-1), Tim-3, and TIGIT. Within all three CD3 + CD4 + CD32 + phenotypes, cells could be identified in infected participants, which contained HIV DNA. CD32 expression on CD4 T cells did not correlate with HIV DNA or cell-associated HIV RNA (both surrogate measures of overall reservoir size) or predict time to rebound viremia following treatment interruption, suggesting that it is not a dominant biomarker for HIV persistence. Our data suggest that while CD32 + T cells can be infected with HIV, CD32 is not a specific marker of the reservoir although it might identify a population of HIV enriched cells in certain situations.

Topical microbicides have the potential to provide effective protection against sexual transmission of HIV. Challenges in developing microbicides include their application in resource-poor settings with high temperatures and a lack of refrigeration, and low user adherence to a rigorous daily regimen. Several protein-based HIV inhibitors show great promise as microbicides, being highly specific and not expected to lead to resistance that would affect the efficacy of current antiretroviral treatments. We show that four potent protein HIV inhibitors, 5P12-RANTES, 5P12-RANTES-L-C37, Grft, and Grft-L-C37 can be formulated into silk fibroin (SF) disks and remain functional for 14 months at 25, 37, and 50 °C. These HIV inhibitor-encapsulated SF disks show excellent inhibition properties in PBMC and in human colorectal and cervical tissue explants, and do not induce inflammatory cytokine secretion. Further, the SF provides a mechanically robust matrix with versatile material formats for this type of application. Finally, a formulation was developed to allow sustained release of functional Grft for 4 weeks at levels sufficient to inhibit HIV transmission. This work establishes the suitability of HIV inhibitor-encapsulated SF disks as topical HIV microbicides that can be further developed to allow easy insertion for extended protection.

To investigate the pharmacokinetics/pharmacodynamics of single-dose maraviroc 300 mg in HIV-1 exposure compartments. Maraviroc concentrations in blood, secretions (vaginal, urethral, oral, and rectal), and tissue (vaginal and rectal) were measured, and ex vivo challenge was performed in 54 healthy volunteers to study protection from HIV infection. Maraviroc Cmax occurred within 4 hours in most compartments. Concentrations from 4 to 72 hours were above intracellular (IC) IC90 in all compartments, range 15-8095 ng/mL. Mean AUC0-72 compartment-to-plasma ratios were highest in the rectum (45-819) and urethra (144) compared with the female genital tract (1.6-4.8) and saliva (0.2). No sex differences in AUC0-72 or Cmax were observed. No ex vivo protection from HIV-1BaL occurred in rectal or vaginal tissue. Despite high and sustained concentrations, single-dose maraviroc was not protective against ex vivo challenge of vaginal/rectal tissue.

Objective: Receptive anal intercourse in both men and women is associated with the highest probability for sexual acquisition of HIV infection. As part of a program to develop an effective prevention strategy, we performed an ex-vivo preclinical evaluation to determine the efficacy of multiple double combinations of maraviroc (MVC) and reverse transcriptase inhibitors (RTIs)., Design: The entry inhibitor, MVC, a nucleotide RTI, tenofovir and two non-nucleoside RTIs, UC781 and TMC120 (dapivirine, DPV), were used in double, combinations against a panel of CCR5-using clade B and clade C HIV-1 isolates and against MVC-escape variants. A gel-formulated version of MVC-DPV combination was also tested., Methods: Indicator cells, cocultures of immature dendritic cells with CD4T cells, and colorectal tissue explants were used to assess antiviral activity of drug combinations., Results: All dual MVC-RTI combinations tested inhibited MVC-sensitive and resistant isolates in cellular and colorectal explants models. All the combinations were positive with no reduction in the activity of MVC. In tissue explants, the combinations against all viral isolates tested produced an increase in the activity of MVC. An initial gel-formulation of MVC-DPV combination showed greater and prolonged antiviral activity of MVC in mucosal tissue explants., Conclusion: This study demonstrates that combinations based on antiretroviral drugs inhibiting HIV transmission at viral entry and reverse transcription have potential as prevention strategies against colorectal transmission of HIV-1 including MVC-resistant isolates. Preclinical evaluation with colorectal tissue explants indicates that a gel-formulation of MVC-DPV is an effective candidate colorectal microbicide.