MAdCAM-1 Co-stimulation Combined with Retinoic Acid and TGF-ß Induces Blood CD8+ T cells to Adopt a Gut CD101+ TRM Phenotype.

Resident memory T cells (TRMs) help control local immune homeostasis and contribute to tissue protective immune responses. The local cues that guide their differentiation and localization are poorly defined. We demonstrate that MAdCAM-1, a ligand for the gut homing receptor α4ß7 integrin, in the presence of retinoic acid and TGF-ß provide a costimulatory signal that induces blood CD8+ T cells to adopt a TRM -like phenotype. These cells express CD103 (integrin αE) and CD69, the two major TRM cell surface markers, along with CD101. They also express CCR5, CCR9 and α4ß7, three receptors associated with gut homing. A subset also express E-cadherin, a ligand for αEß7. Fluorescent lifetime imaging indicated an αEß7 and E-cadherin cis interaction on the plasma membrane. This report advances our understanding of the signals that drive the differentiation of CD8+ T cells into TRMs and provides a means to expand these cells in vitro, thereby affording an avenue to generate more effective tissue specific immunotherapies.

TGF-ß blockade drives a transitional effector phenotype in T cells reversing SIV latency and decreasing SIV reservoirs in vivo.

HIV-1 persistence during ART is due to the establishment of long-lived viral reservoirs in resting immune cells. Using an NHP model of barcoded SIVmac239 intravenous infection and therapeutic dosing of anti-TGFBR1 inhibitor galunisertib (LY2157299), we confirm the latency reversal properties of in vivo TGF-ß blockade, decrease viral reservoirs and stimulate immune responses. Treatment of eight female, SIV-infected macaques on ART with four 2-weeks cycles of galunisertib leads to viral reactivation as indicated by plasma viral load and immunoPET/CT with a 64Cu-DOTA-F(ab')2-p7D3-probe. Post-galunisertib, lymph nodes, gut and PBMC exhibit lower cell-associated (CA-)SIV DNA and lower intact pro-virus (PBMC). Galunisertib does not lead to systemic increase in inflammatory cytokines. High-dimensional cytometry, bulk, and single-cell (sc)RNAseq reveal a galunisertib-driven shift toward an effector phenotype in T and NK cells characterized by a progressive downregulation in TCF1. In summary, we demonstrate that galunisertib, a clinical stage TGF-ß inhibitor, reverses SIV latency and decreases SIV reservoirs by driving T cells toward an effector phenotype, enhancing immune responses in vivo in absence of toxicity.

The V2 domain of HIV gp120 mimics an interaction between CD4 and integrin ⍺4ß7.

The CD4 receptor, by stabilizing TCR-MHC II interactions, plays a central role in adaptive immunity. It also serves as the HIV docking receptor. The HIV gp120 envelope protein binds directly to CD4. This interaction is a prerequisite for viral entry. gp120 also binds to ⍺4ß7, an integrin that is expressed on a subset of memory CD4+ T cells. HIV tropisms for CD4+ T cells and gut tissues are central features of HIV pathogenesis. We report that CD4 binds directly to ⍺4ß7 in a dynamic way, consistent with a cis regulatory interaction. The molecular details of this interaction are related to the way in which gp120 interacts with both receptors. Like MAdCAM-1 and VCAM-1, two recognized ligands of ⍺4ß7, the binding interface on CD4 includes 2 sites (1° and accessory), distributed across its two N-terminal IgSF domains (D1 and D2). The 1° site includes a sequence in the G ß-strand of CD4 D2, KIDIV, that binds directly to ⍺4ß7. This pentapeptide sequence occurs infrequently in eukaryotic proteins. However, a closely related and conserved sequence, KLDIV, appears in the V2 domain of gp120. KLDIV mediates gp120-⍺4ß7 binding. The accessory ⍺4ß7 binding site on CD4 includes Phe43. The Phe43 aromatic ring protrudes outward from one edge of a loop connecting the C'C" strands of CD4 D1. Phe43 is a principal contact for HIV gp120. It interacts with conserved residues in the recessed CD4 binding pocket. Substitution of Phe43 abrogates CD4 binding to both gp120 and ⍺4ß7. As such, the interactions of gp120 with both CD4 and ⍺4ß7 reflect elements of their interactions with each other. These findings indicate that gp120 specificities for CD4 and ⍺4ß7 are interrelated and suggest that selective pressures which produced a CD4 tropic virus that replicates in gut tissues are linked to a dynamic interaction between these two receptors.

Herpes Simplex Virus Type 2 Prevalence and Association with Inflammatory Cytokines Among Sexual and Gender Minorities Living With and Without HIV-1 from Lagos, Nigeria.

Herpes simplex virus type 2 (HSV-2) is common globally and contributes significantly to the risk of acquiring HIV-1, yet these two sexually transmitted infections have not been sufficiently characterized for sexual and gender minorities (SGM) across Sub-Saharan Africa. To help fill this gap, we performed a retrospective study using plasma and serum samples from 183 SGM enrolled at the Lagos site of the TRUST/RV368 cohort in Nigeria, assayed them for HSV-2 antibodies with the Kalon ELISA and plasma cytokines and chemokines with Luminex, and correlated the findings with HIV-1 viral loads (VLs) and CD4 counts. We found an overall HSV-2 prevalence of 36.6% (49.5% and 23.9% among SGM with and without HIV-1, respectively, p < .001). Moreover, HSV-2-positive status was associated with high circulating concentrations of CCL11 among antiretroviral therapy-treated (p = .031) and untreated (p = .015) participants, and with high concentrations of CCL2 in the untreated group (p = .004), independent of VL. Principal component analysis revealed a strong association of cytokines with HIV-1 VL independent of HSV-2 status. In conclusion, our study finds that HSV-2 prevalence among SGM with HIV-1 is twice as high than HSV-2 prevalence among SGM without HIV-1 in Lagos and suggests that this is associated with higher levels of certain systemic cytokines. Additional work is needed to further characterize the relationship between HSV-2 and HIV-1 in SGM and help develop targeted therapies for coinfected individuals.

A multicenter randomized trial for quality of life evaluation by non-invasive intelligent tools during post-curative treatment follow-up for head and neck cancer: Clinical study protocol.

Patients surviving head and neck cancer (HNC) suffer from high physical, psychological, and socioeconomic burdens. Achieving cancer-free survival with an optimal quality of life (QoL) is the primary goal for HNC patient management. So, maintaining lifelong surveillance is critical. An ambitious goal would be to carry this out through the advanced analysis of environmental, emotional, and behavioral data unobtrusively collected from mobile devices. The aim of this clinical trial is to reduce, with non-invasive tools (i.e., patients' mobile devices), the proportion of HNC survivors (i.e., having completed their curative treatment from 3 months to 10 years) experiencing a clinically relevant reduction in QoL during follow-up. The Big Data for Quality of Life (BD4QoL) study is an international, multicenter, randomized (2:1), open-label trial. The primary endpoint is a clinically relevant global health-related EORTC QLQ-C30 QoL deterioration (decrease ≥10 points) at any point during 24 months post-treatment follow-up. The target sample size is 420 patients. Patients will be randomized to be followed up using the BD4QoL platform or per standard clinical practice. The BD4QoL platform includes a set of services to allow patients monitoring and empowerment through two main tools: a mobile application installed on participants' smartphones, that includes a chatbot for e-coaching, and the Point of Care dashboard, to let the investigators manage patients data. In both arms, participants will be asked to complete QoL questionnaires at study entry and once every 6 months, and will undergo post-treatment follow up as per clinical practice. Patients randomized to the intervention arm (n=280) will receive access to the BD4QoL platform, those in the control arm (n=140) will not. Eligibility criteria include completing curative treatments for non-metastatic HNC and the use of an Android-based smartphone. Patients undergoing active treatments or with synchronous cancers are excluded. Clinical Trial Registration: ClinicalTrials.gov, identifier (NCT05315570).

Blockade of TGF-ß signaling reactivates HIV-1/SIV reservoirs and immune responses in vivo.

TGF-ß plays a critical role in maintaining immune cells in a resting state by inhibiting cell activation and proliferation. Resting HIV-1 target cells represent the main cellular reservoir after long-term antiretroviral therapy (ART). We hypothesized that releasing cells from TGF-ß-driven signaling would promote latency reversal. To test our hypothesis, we compared HIV-1 latency models with and without TGF-ß and a TGF-ß type 1 receptor inhibitor, galunisertib. We tested the effect of galunisertib in SIV-infected, ART-treated macaques by monitoring SIV-env expression via PET/CT using the 64Cu-DOTA-F(ab')2 p7D3 probe, along with plasma and tissue viral loads (VLs). Exogenous TGF-ß reduced HIV-1 reactivation in U1 and ACH-2 models. Galunisertib increased HIV-1 latency reversal ex vivo and in PBMCs from HIV-1-infected, ART-treated, aviremic donors. In vivo, oral galunisertib promoted increased total standardized uptake values in PET/CT images in gut and lymph nodes of 5 out of 7 aviremic, long-term ART-treated, SIV-infected macaques. This increase correlated with an increase in SIV RNA in the gut. Two of the 7 animals also exhibited increases in plasma VLs. Higher anti-SIV T cell responses and antibody titers were detected after galunisertib treatment. In summary, our data suggest that blocking TGF-ß signaling simultaneously increases retroviral reactivation events and enhances anti-SIV immune responses.

An immunoPET probe to SARS-CoV-2 reveals early infection of the male genital tract in rhesus macaques.

The systemic nature of SARS-CoV-2 infection is highly recognized, but poorly characterized. A non-invasive and unbiased method is needed to clarify whole body spatiotemporal dynamics of SARS-CoV-2 infection after transmission. We recently developed a probe based on the anti-SARS-CoV-2 spike antibody CR3022 to study SARS-CoV-2 pathogenesis in vivo. Herein, we describe its use in immunoPET to investigate SARS-CoV-2 infection of three rhesus macaques. Using PET/CT imaging of macaques at different times post-SARS-CoV-2 inoculation, we track the 64Cu-labelled CR3022-F(ab')2 probe targeting the spike protein of SARS-CoV-2 to study the dynamics of infection within the respiratory tract and uncover novel sites of infection. Using this method, we uncovered differences in lung pathology between infection with the WA1 isolate and the delta variant, which were readily corroborated through computed tomography scans. The 64Cu-CR3022-probe also demonstrated dynamic changes occurring between 1- and 2-weeks post-infection. Remarkably, a robust signal was seen in the male genital tract (MGT) of all three animals studied. Infection of the MGT was validated by immunofluorescence imaging of infected cells in the testicular and penile tissue and severe pathology was observed in the testes of one animal at 2-weeks post-infection. The results presented here underscore the utility of using immunoPET to study the dynamics of SARS-CoV-2 infection to understand its pathogenicity and discover new anatomical sites of viral replication. We provide direct evidence for SARS-CoV-2 infection of the MGT in rhesus macaques revealing the possible pathologic outcomes of viral replication at these sites.

An immunoPET probe to SARS-CoV-2 reveals early infection of the male genital tract in rhesus macaques.

The systemic nature of SARS-CoV-2 infection is highly recognized, but poorly characterized. A non-invasive and unbiased method is needed to clarify whole body spatiotemporal dynamics of SARS-CoV-2 infection after transmission. We recently developed a probe based on the anti-SARS-CoV-2 spike antibody CR3022 to study SARS-CoV-2 pathogenesis in vivo. Herein, we describe its use in immunoPET to investigate SARS-CoV-2 infection of three rhesus macaques. Using PET/CT imaging of macaques at different times post-SARS-CoV-2 inoculation, we track the 64Cu-labelled CR3022-F(ab')2 probe targeting the spike protein of SARS-CoV-2 to study the dynamics of infection within the respiratory tract and uncover novel sites of infection. Using this method, we uncovered differences in lung pathology between infection with the WA1 isolate and the delta variant, which were readily corroborated through computed tomography scans. The 64Cu-CR3022-probe also demonstrated dynamic changes occurring between 1- and 2-weeks post-infection. Remarkably, a robust signal was seen in the male genital tract (MGT) of all three animals studied. Infection of the MGT was validated by immunofluorescence imaging of infected cells in the testicular and penile tissue and severe pathology was observed in the testes of one animal at 2-weeks post-infection. The results presented here underscore the utility of using immunoPET to study the dynamics of SARS-CoV-2 infection to understand its pathogenicity and discover new anatomical sites of viral replication. We provide direct evidence for SARS-CoV-2 infection of the MGT in rhesus macaques revealing the possible pathologic outcomes of viral replication at these sites.

HIV-1 Establishes a Sanctuary Site in the Testis by Permeating the BTB Through Changes in Cytoskeletal Organization.

Studies suggest that HIV-1 invades the testis through initial permeation of the blood-testis barrier (BTB). The selectivity of the BTB to antiretroviral drugs makes this site a sanctuary for the virus. Little is known about how HIV-1 crosses the BTB and invades the testis. Herein, we used 2 approaches to examine the underlying mechanism(s) by which HIV-1 permeates the BTB and gains entry into the seminiferous epithelium. First, we examined if recombinant Tat protein was capable of perturbing the BTB and making the barrier leaky, using the primary rat Sertoli cell in vitro model that mimics the BTB in vivo. Second, we used HIV-1-infected Sup-T1 cells to investigate the activity of HIV-1 infection on cocultured Sertoli cells. Using both approaches, we found that the Sertoli cell tight junction permeability barrier was considerably perturbed and that HIV-1 effectively permeates the BTB by inducing actin-, microtubule-, vimentin-, and septin-based cytoskeletal changes in Sertoli cells. These studies suggest that HIV-1 directly perturbs BTB function, potentially through the activity of the Tat protein.

Blocking α4ß7 integrin delays viral rebound in SHIVSF162P3-infected macaques treated with anti-HIV broadly neutralizing antibodies.

Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin α4ß7 with an anti-α4ß7 monoclonal antibody (Rh-α4ß7) affects immune responses in SIV/SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with α4ß7 integrin blockade, SHIVSF162P3-infected, viremic rhesus macaques were treated with bNAbs only (VRC07-523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-α4ß7 or were left untreated as a control. Treatment with bNAbs alone decreased viremia below 200 copies/ml in all macaques, but seven of eight macaques (87.5%) in the bNAbs-only group rebounded within a median of 3 weeks (95% CI: 2 to 9). In contrast, three of six macaques treated with a combination of Rh-α4ß7 and bNAbs (50%) maintained a viremia below 200 copies/ml until the end of the follow-up period; viremia in the other three macaques rebounded within a median of 6 weeks (95% CI: 5 to 11). Thus, there was a modest delay in viral rebound in the macaques treated with the combination antibody therapy compared to bNAbs alone. Our study suggests that α4ß7 integrin blockade may prolong virologic control by bNAbs in SHIVSF162P3-infected macaques.

The Vaginal Microbiota, Bacterial Biofilms and Polymeric Drug-Releasing Vaginal Rings.

The diversity and dynamics of the microbial species populating the human vagina are increasingly understood to play a pivotal role in vaginal health. However, our knowledge about the potential interactions between the vaginal microbiota and vaginally administered drug delivery systems is still rather limited. Several drug-releasing vaginal ring products are currently marketed for hormonal contraception and estrogen replacement therapy, and many others are in preclinical and clinical development for these and other clinical indications. As with all implantable polymeric devices, drug-releasing vaginal rings are subject to surface bacterial adherence and biofilm formation, mostly associated with endogenous microorganisms present in the vagina. Despite more than 50 years since the vaginal ring concept was first described, there has been only limited study and reporting around bacterial adherence and biofilm formation on rings. With increasing interest in the vaginal microbiome and vaginal ring technology, this timely review article provides an overview of: (i) the vaginal microbiota, (ii) biofilm formation in the human vagina and its potential role in vaginal dysbiosis, (iii) mechanistic aspects of biofilm formation on polymeric surfaces, (iv) polymeric materials used in the manufacture of vaginal rings, (v) surface morphology characteristics of rings, (vi) biomass accumulation and biofilm formation on vaginal rings, and (vii) regulatory considerations.

Outcomes in Living Donor Kidney Transplantation: The Role of Donor's Kidney Function.

INTRODUCTION: Living donor kidney transplant (LDKT) is one of the best therapeutic options for end-stage kidney disease (ESKD). Guidelines identify different estimated glomerular filtration rate (eGFR) thresholds to determine the eligibility of donors. The aim of our study was to evaluate whether pretransplant donor eGFR was associated with kidney function in the recipient. METHODS: We retrospectively studied LDKT recipients who received a kidney graft between September 1, 2005, and June 30, 2016 in the same transplant center in France and that had eGFR data available at 3, 12, 24, and 36 months posttransplant. RESULTS: We studied 90 donor-recipient pairs. The average age at time of transplant was 51.47 ± 10.95 for donors and 43.04 ± 13.52 years for recipients. Donors' average eGFR was 91.99 ± 15.37 mL/min/1.73 m2. Donor's age and eGFR were significantly correlated (p < 0.0001, r2 0.023). Donor's age and eGFR significantly correlated with recipient's eGFR at 3, 12, and 24 months posttransplant (age: p < 0.001 at all intervals; eGFR p = 0.001, 0.003, and 0.016, respectively); at 36 months, only donor's age significantly correlated with recipient's eGFR. BMI, gender match, and year of kidney transplant did not correlate with graft function. In the multivariable analyses, donor's eGFR and donor's age were found to be associated with graft function; correlation with eGFR was lost at 36 months; and donor's age retained a strong correlation with graft function at all intervals (p < 0.001). CONCLUSIONS: Donor's eGFR and age are strong predictors of recipient's kidney function at 3 years. We suggest that donor's eGFR should be clinically balanced with other determinants of kidney function and in particular with age.

Development of an In Vivo Probe to Track SARS-CoV-2 Infection in Rhesus Macaques.

Infection with the novel coronavirus, SARS-CoV-2, results in pneumonia and other respiratory symptoms as well as pathologies at diverse anatomical sites. An outstanding question is whether these diverse pathologies are due to replication of the virus in these anatomical compartments and how and when the virus reaches those sites. To answer these outstanding questions and study the spatiotemporal dynamics of SARS-CoV-2 infection a method for tracking viral spread in vivo is needed. We developed a novel, fluorescently labeled, antibody-based in vivo probe system using the anti-spike monoclonal antibody CR3022 and demonstrated that it could successfully identify sites of SARS-CoV-2 infection in a rhesus macaque model of COVID-19. Our results showed that the fluorescent signal from our antibody-based probe could differentiate whole lungs of macaques infected for 9 days from those infected for 2 or 3 days. Additionally, the probe signal corroborated the frequency and density of infected cells in individual tissue blocks from infected macaques. These results provide proof of concept for the use of in vivo antibody-based probes to study SARS-CoV-2 infection dynamics in rhesus macaques.

Anti-α4ß7 monoclonal antibody-conjugated nanoparticles block integrin α4ß7 on intravaginal T cells in rhesus macaques.

Intravenous administration of anti-α4ß7 monoclonal antibody in macaques decreases simian immunodeficiency virus (SIV) vaginal infection and reduces gut SIV loads. Because of potential side effects of systemic administration, a prophylactic strategy based on mucosal administration of anti-α4ß7 antibody may be safer and more effective. With this in mind, we developed a novel intravaginal formulation consisting of anti-α4ß7 monoclonal antibody-conjugated nanoparticles (NPs) loaded in a 1% hydroxyethylcellulose (HEC) gel (NP-α4ß7 gel). When intravaginally administered as a single dose in a rhesus macaque model, the formulation preferentially bound to CD4+ or CD3+ T cells expressing high levels of α4ß7, and occupied ~40% of α4ß7 expressed by these subsets and ~25% of all cells expressing α4ß7 Blocking of the α4ß7 was restricted to the vaginal tract without any changes detected systemically.

A Tat/Rev Induced Limiting Dilution Assay to Measure Viral Reservoirs in Non-Human Primate Models of HIV Infection.

The establishment of latent infection and poorly characterized viral reservoirs in tissues represent major obstacles to a definitive cure for HIV. Non-human primate (NHP) models of HIV infection are critical to elucidate pathogenic processes and an essential tool to test novel therapeutic strategies. Thus, the availability of novel assays to measure residual viral replication and reservoirs in NHP models may increase their utility in the search for an HIV cure. We developed a tat/rev induced limiting dilution assay to measure the frequency of CD4+ T cells that express multiply-spliced(ms)_SIV RNA in presence and absence of stimulation. We validated the assay using cell lines and cells from blood and lymph nodes of SIV infected macaques. In vitro, SIV/SHIV TILDA detects only cells expressing viral proteins. In SIV/SHIV-infected macaques, CD4+ T cells that express msSIV/SHIV RNA (TILDA data) were detected also in the setting of very low/undetectable viremia. TILDA data were significantly higher after stimulation and correlated with plasma viral load (pVL). Interestingly, TILDA data from early cART initiation correlated with peak and AUC pVL post-cART interruption. In summary, we developed an assay that may be useful in characterizing viral reservoirs and determining the effect of HIV interventions in NHP models.

Delayed vaginal SHIV infection in VRC01 and anti-α4ß7 treated rhesus macaques.

VRC01 protects macaques from vaginal SHIV infection after a single high-dose challenge. Infusion of a simianized anti-α4ß7 mAb (Rh-α4ß7) just prior to, and during repeated vaginal exposures to SIVmac251 partially protected macaques from vaginal SIV infection and rescued CD4+ T cells. To investigate the impact of combining VRC01 and Rh-α4ß7 on SHIV infection, 3 groups of macaques were treated with a suboptimal dosing of VRC01 alone or in combination with Rh-α4ß7 or with control antibodies prior to the initiation of weekly vaginal exposures to a high dose (1000 TCID50) of SHIVAD8-EO. The combination Rh-α4ß7-VRC01 significantly delayed SHIVAD8-EO vaginal infection. Following infection, VRC01-Rh-α4ß7-treated macaques maintained higher CD4+ T cell counts and exhibited lower rectal SIV-DNA loads compared to controls. Interestingly, VRC01-Rh-α4ß7-treated macaques had fewer IL-17-producing cells in the blood and the gut during the acute phase of infection. Moreover, higher T cell responses to the V2-loop of the SHIVAD8-EO envelope in the VRC01-Rh-α4ß7 group inversely correlated with set point viremia. The combination of suboptimal amounts of VRC01 and Rh-α4ß7 delayed infection, altered antiviral immune responses and minimized CD4+ T cell loss. Further exploration of the effect of combining bNAbs with Rh-α4ß7 on SIV/HIV infection and antiviral immune responses is warranted and may lead to novel preventive and therapeutic strategies.

Mucosal Susceptibility to Human Immunodeficiency Virus Infection in the Proliferative and Secretory Phases of the Menstrual Cycle.

Factors underlying HIV acquisition in women remain incompletely understood. This study evaluated ex vivo mucosal HIV-1BaL infection (ectocervix, endocervix), T cell frequencies and phenotype (ectocervix, endocervix, peripheral blood), and HIV-1BaL-induced tissue immune responses (ectocervix) in the proliferative and secretory phases of the menstrual cycle using samples obtained from women undergoing hysterectomies. Tissue infectivity (number of productively infected explants) and infection level following 500 and/or fifty 50% tissue culture infectious dose (TCID50) HIV-1BaL challenge were similar in the proliferative and secretory phases. Although not associated with infection outcomes, higher frequencies of HIV target CD4+α4ß7+ T cells, and stronger HIV-1BaL-induced proinflammatory responses were detected in ectocervix in the secretory versus proliferative phase. Independently of the cycle phase, serum E2 concentrations were inversely associated with ectocervical and endocervical tissue infection levels following high-dose 500 TCID50 HIV-1BaL challenge, with frequencies of CD4+α4ß7+ T cells in endocervix, and with HIV-induced interleukin (IL)2R and IL4 in ectocervix. Although serum P4 concentrations and P4/E2 ratios were neither associated with tissue infection level nor infectivity, high P4 concentrations and/or P4/E2 ratios correlated with high frequencies of CD4+α4ß7+ T cells in ectocervix, low frequencies of CD4+CD103+ blood T cells, low CD4+LFA-1+ T cells in endocervix, and high proinflammatory (IL1ß, IL17, tumor necrosis factor α) ectocervical tissue responses to HIV-1BaL. The data suggest an inhibitory effect of E2 on mucosal HIV infection, provide insights into potential mechanisms of E2-mediated anti-HIV activity, and highlight P4-associated immune changes in the mucosa.

Griffithsin carrageenan fast dissolving inserts prevent SHIV HSV-2 and HPV infections in vivo.

Human immunodeficiency virus (HIV) pre-exposure prophylaxis (PrEP) strategies with proven in vivo efficacy rely on antiretroviral drugs, creating the potential for drug resistance and complicated treatment options in individuals who become infected. Moreover, on-demand products are currently missing from the PrEP development portfolio. Griffithsin (GRFT) is a non-antiretroviral HIV entry inhibitor derived from red algae with an excellent safety profile and potent activity in vitro. When combined with carrageenan (CG), GRFT has strong activity against herpes simplex virus-2 (HSV-2) and human papillomavirus (HPV) in vitro and in vivo. Here, we report that GRFT/CG in a freeze-dried fast dissolving insert (FDI) formulation for on-demand use protects rhesus macaques from a high dose vaginal SHIV SF162P3 challenge 4 h after FDI insertion. Furthermore, the GRFT/CG FDI also protects mice vaginally against HSV-2 and HPV pseudovirus. As a safe, potent, broad-spectrum, on-demand non-antiretroviral product, the GRFT/CG FDI warrants clinical development.