Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques.

The human immunodeficiency virus epidemic continues in sub-Saharan Africa, and particularly affects adolescent girls and women who have limited access to antiretroviral therapy. Here we report that the risk of vaginal simian immunodeficiency virus (SIV)mac251 acquisition is reduced by more than 90% using a combination of a vaccine comprising V1-deleted (V2 enhanced) SIV envelope immunogens with topical treatment of the zinc-finger inhibitor SAMT-247. Following 14 weekly intravaginal exposures to the highly pathogenic SIVmac251, 80% of a cohort of 20 macaques vaccinated and treated with SAMT-247 remained uninfected. In an arm of 18 vaccinated-only animals without microbicide, 40% of macaques remained uninfected. The combined SAMT-247/vaccine regimen was significantly more effective than vaccination alone. By analysing immune correlates of protection, we show that, by increasing zinc availability, SAMT-247 increases natural killer cytotoxicity and monocyte efferocytosis, and decreases T-cell activation to augment vaccine-induced protection.

Direct intranodal tonsil vaccination with modified vaccinia Ankara vaccine protects macaques from highly pathogenic SIVmac251.

Human immunodeficiency virus (HIV) is a mucosally transmitted virus that causes immunodeficiency and AIDS. Developing efficacious vaccines to prevent infection is essential to control the epidemic. Protecting the vaginal and rectal mucosa, the primary routes of HIV entry has been a challenge given the significant compartmentalization between the mucosal and peripheral immune systems. We hypothesized that direct intranodal vaccination of mucosa associated lymphoid tissue (MALT) such as the readily accessible palatine tonsils could overcome this compartmentalization. Here we show that rhesus macaques primed with plasmid DNA encoding SIVmac251-env and gag genes followed by an intranodal tonsil MALT boost with MVA encoding the same genes protects from a repeated low dose intrarectal challenge with highly pathogenic SIVmac251; 43% (3/7) of vaccinated macaques remained uninfected after 9 challenges as compared to the unvaccinated control (0/6) animals. One vaccinated animal remained free of infection even after 22 challenges. Vaccination was associated with a ~2 log decrease in acute viremia that inversely correlated with anamnestic immune responses. Our results suggest that a combination of systemic and intranodal tonsil MALT vaccination could induce robust adaptive and innate immune responses leading to protection from mucosal infection with highly pathogenic HIV and rapidly control viral breakthroughs.

E4orf1 Suppresses E1B-Deleted Adenovirus Vaccine-Induced Immune Responses.

As demonstrated by the recent COVID pandemic, vaccines can reduce the burden arising from infectious agents. Adenoviruses (Ads) with deletion of the early region 1B55K (ΔE1B Ad) are currently being explored for use in vaccine delivery. ΔE1B Ads are different from Ads with deletions in early region 1 and early region 3 (ΔE1/E3) used in most Ad vaccine vectors in that they contain the Ad early region 1A (E1A), and therefore the ability to replicate. Common to almost all Ads that are being explored for clinical use is the Ad early region 4 (E4). Among the E4 genes is open reading frame 1 (E4orf1), which mediates signals through the PI3-kinase/Akt pathway that is known to modulate immune responses. This suggests that E4orf1 might also modulate immune responses, although it has remained unexplored in ΔE1B Ad. Here, we show that cells infected with an E1B55K and E4orf1-deleted (ΔE41) Ad exhibited reduced levels of phosphorylated Akt (Ser473 and Thr308)) and expressed different intrinsic innate immune cytokines from those induced in cells infected with an E4orf1-containing, ΔE1B parental Ad that exhibited elevated levels of phosphorylated Akt. Rhesus macaques immunized with a ΔE41 Ad that expressed rhFLSC (HIV-1BaL gp120 linked to rhesus CD4 D1 and D2), exhibited higher levels of rhFLSC-specific interferon γ-producing memory T-cells, higher titers of rhFLSC-specific IgG1 binding antibody in serum, and antibodies able to mediate antibody-dependent cellular cytotoxicity (ADCC) with greater killing capacity than the ΔE1B Ad. Therefore, E4orf1, perhaps by acting through the PI3-kinase/Akt pathway, limits intrinsic innate and system-wide adaptive immune responses that are important for improved ΔE1B Ad-based vaccines.

Disruption of NBS1/MRN Complex Formation by E4orf3 Supports NF-κB That Licenses E1B55K-Deleted Adenovirus-Infected Cells to Accumulate DNA>4n.

Cells increase their DNA content greater than the G2/M (DNA > 4n) phases along the path to cancer. The signals that support this increase in DNA content remain poorly understood. Cells infected with adenovirus (Ad) similarly develop DNA > 4n and share a need to bypass the DNA damage response (DDR) signals that trigger cell cycle arrest, and/or cell death. Ads with deletion in early region 1B55K (ΔE1B Ad) are oncolytic agents that are currently being explored for use in vaccine delivery. Interestingly, they promote higher levels of DNA > 4n than Ads that contain E1B55K. Existing in these and almost all Ads that are being explored for clinical use, is early region 4 (E4). The Ad E4 open reading frame 3 (E4orf3) is a viral oncogene that interferes with the ability of cells to respond to DNA damage by disrupting MRN complex formation. Our study reveals that E4orf3 is required for the enhanced fraction of ΔE1B Ad-infected cells with DNA > 4n. For that reason, we explored signaling events mediated by E4orf3. We found that in ΔE1B Ad-infected cells, E4orf3, as reported by others, isolates NBS1 in nuclear dots and tracks. This allows for elevated levels of phosphorylated ATM that is linked to transcriptionally active NF-κB. Pharmacological inhibition of NF-κB reduced the fraction of ΔE1B Ad-infected cells with DNA > 4n while pharmacological inhibition of ATM reduced the levels of nuclear NF-κB and the fraction of ΔE1B Ad-infected cells with DNA > 4n and increased the fraction of dead or dying cells with fragmented DNA. This ability of E4orf3 to disrupt MRN complex formation that allows cells to bypass the cell cycle, evade death, and accumulate DNA > 4n, may be linked to its oncogenic potential. IMPORTANCE Genome instability, a hallmark of cancer, exists as part of a cycle that leads to DNA damage and DNA > 4n that further enhances genome instability. Ad E4orf3 is a viral oncogene. Here, we describe E4orf3 mediated signaling events that support DNA > 4n in ΔE1B Ad-infected cells. These signaling events may be linked to the oncogenic potential of E4orf3 and may provide a basis for how some cells survive with DNA > 4n.

Virus Control in Vaccinated Rhesus Macaques Is Associated with Neutralizing and Capturing Antibodies against the SHIV Challenge Virus but Not with V1V2 Vaccine-Induced Anti-V2 Antibodies Alone.

The role of vaccine-induced anti-V2 Abs was tested in three protection experiments in rhesus macaques. In an experiment using immunogens similar to those in the RV144 vaccine trial (Anti-envelope [Env]), nine rhesus macaques were coimmunized with gp16092TH023 DNA and SIV gag and gp120A244 and gp120MN proteins. In two V2-focused experiments (Anti-V2 and Anti-V2 Mucosal), nine macaques in each group were immunized with V1V292TH023 DNA, V1V2A244 and V1V2CasaeA2 proteins, and cyclic V2CaseA2 peptide. DNA and protein immunogens, formulated in Adjuplex, were given at 0, 4, 12, and 20 weeks, followed by intrarectal SHIVBaL.P4 challenges. Peak plasma viral loads (PVL) of 106-107 copies/ml developed in all nine sham controls. Overall, PVL was undetectable in one third of immunized macaques, and two animals tightly controlled the virus with the Anti-V2 Mucosal vaccine strategy. In the Anti-Env study, Abs that captured or neutralized SHIVBaL.P4 inversely correlated with PVL. Conversely, no correlation with PVL was found in the Anti-V2 experiments with nonneutralizing plasma Abs that only captured virus weakly. Titers of Abs against eight V1V2 scaffolds and cyclic V2 peptides were comparable between controllers and noncontrollers as were Ab-dependent cellular cytotoxicity and Ab-dependent cell-mediated virus inhibition activities against SHIV-infected target cells and phagocytosis of gp120-coated beads. The Anti-Env experiment supports the role of vaccine-elicited neutralizing and nonneutralizing Abs in control of PVL. However, the two V2-focused experiments did not support a role for nonneutralizing V2 Abs alone in controlling PVL, as neither Ab-dependent cellular cytotoxicity, Ab-dependent cell-mediated virus inhibition, nor phagocytosis correlated inversely with heterologous SHIVBaL.P4 infection.

The immunological impact of adenovirus early genes on vaccine-induced responses in mice and nonhuman primates.

Adenovirus (Ad) is being explored for use in the prevention and treatment of a variety of infectious diseases and cancers. Ad with a deletion in early region 3 (ΔE3) provokes a stronger immune response than Ad with deletions in early regions 1 and E3 (ΔE1/ΔE3). The ΔE1/ΔE3 Ads are more popular because they can carry a larger transgene and because of the deleted E1 (E1A and E1B), are perceived safer for clinical use. Ad with a deletion in E1B55K (ΔE1B55K) has been in phase III clinical trials for use in cancer therapy in the US and has been approved for use in head and neck tumor therapy in China, demonstrating that Ad containing E1A are safe for clinical use. We have shown previously that ΔE1B55K Ad, even while promoting lower levels of an inserted transgene, promoted similar levels of transgene-specific immune responses as a ΔE3 Ad. Products of the Ad early region 4 (E4) limit the ability of cells to mount an innate immune response. Using this knowledge, we deleted the Ad E4 open reading frames 1-4 (E4orf1-4) from the ΔE1B55K Ad. Here, we show that innate cytokine network genes are elevated in the ΔE4 Ad-infected cells beyond that of ΔE3 Ad-infected cells. Further, in immunized mice the IgG2a subclass was favored as was the IgG1 subclass in immunized nonhuman primates. Thus, Ad E4 impacts immune responses in cells, in immunized mice, and immunized nonhuman primates. These Ad may offer advantages that are beneficial for clinical use.Importance: Adenovirus (Ad) is being explored for use in the prevention and treatment of a variety of infectious diseases and cancers. Here we provide evidence in cells, mice, and nonhuman primates supporting the notion that Ad early gene-products limit specific immune responses. Ad constructed with deletions in early genes and expressing HIV envelope protein was shown to induce greater HIV-specific cellular immune responses and higher titer antibodies compared to the parental Ad with the early genes. In addition to eliciting enhanced immunity, the deleted Ad possesses more space for insertion of additional or larger transgenes needed for targeting other infectious agents or cancers.

A Mucosal Adenovirus Prime/Systemic Envelope Boost Vaccine Regimen Elicits Responses in Cervicovaginal and Alveolar Macrophages of Rhesus Macaques Associated With Delayed SIV Acquisition and B Cell Help.

Vaccine strategies targeting the mucosal portal of entry may prevent HIV acquisition and systemic infection. Macrophages in cervicovaginal compartments are one of the first cell types to encounter virus upon vaginal exposure. Their activation can lead to recruitment of additional macrophages and CD4+ T-cells susceptible to viral infection. However, they are also critical in providing early protection against invading pathogens. Therefore, understanding their response to immunization is important for vaccine design. We immunized rhesus macaques twice mucosally with replicating adenovirus (Ad) SIV recombinants, followed by two intramuscular boosts with SIV gp120 protein. Macaques were subsequently challenged intravaginally with repeated low doses of SIVmac251. Using flow cytometry, we evaluated responses of cervicovaginal macrophages (CVM) and alveolar macrophages (AM) in bronchoalveolar lavage as initial immunization was to the upper respiratory tract. The frequency of CVM increased over the course of immunization; however, CCR5 expression significantly decreased. Significantly increased expression of the chemokines CCL3 (p < 0.01), CCL4, CCL5, and CXCL8 (p < 0.0001 for all) on CVM was seen post-1st Ad but their expression significantly decreased post-2nd boost. CD4+ T-cell frequency in the cervical mucosa remained unchanged. CVM FcγRIII expression was significantly increased at all time points post-immunization compared to naïve animals. FcγRIII expression post-2nd Ad positively correlated with the number of challenges needed for infection (r = 0.68; p = 0.0051). Vaccination increased AM FcγRIII expression which post-2nd boost correlated with antibody-dependent phagocytosis. Activation of AMs was evident by increased expression of CD40 and CD80 post-2nd Ad compared to naïve macaques. APRIL expression also significantly increased post-2nd Ad and correlated with B cell frequency in bronchoalveolar lavage (BAL) (r = 0.73; p = 0.0019) and total IgG in BAL-fluid (r = 0.53; p = 0.047). B cells cultured with SIV gp120-stimulated AM supernatant from vaccinated macaques exhibited significant increases in B cell activation markers CD38 and CD69 compared to B cells cultured alone or with AM supernatant from unvaccinated macaques. Overall, the vaccine regimen did not induce recruitment of susceptible cells to the vaginal mucosa but increased CVM FcγRIII expression which correlated with delayed SIV acquisition. Further, immunization induced expression of AM cytokines, including those associated with providing B cell help.

A Prime/Boost Vaccine Regimen Alters the Rectal Microbiome and Impacts Immune Responses and Viremia Control Post-Simian Immunodeficiency Virus Infection in Male and Female Rhesus Macaques.

An efficacious human immunodeficiency virus (HIV) vaccine will likely require induction of both mucosal and systemic immune responses. We compared the immunogenicity and protective efficacy of two mucosal/systemic vaccine regimens and investigated their effects on the rectal microbiome. Rhesus macaques were primed twice mucosally with replication-competent adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus (SIV) recombinants and boosted twice intramuscularly with ALVAC-SIV recombinant plus SIV gp120 protein or with DNA for SIV genes and rhesus interleukin-12 plus SIV gp120 protein. Controls received empty Ad5hr vector and alum adjuvant only. Both regimens elicited strong, comparable mucosal and systemic cellular and humoral immunity. Prevaccination rectal microbiomes of males and females differed and significantly changed over the course of immunization, most strongly in females after Ad5hr immunizations. Following repeated low-dose intrarectal SIV challenges, both vaccine groups exhibited modestly but significantly reduced acute viremia. Male and female controls exhibited similar acute viral loads; however, vaccinated females, but not males, exhibited lower levels of acute viremia, compared to same-sex controls. Few differences in adaptive immune responses were observed between the sexes. Striking differences in correlations of the rectal microbiome of males and females with acute viremia and immune responses associated with protection were seen and point to effects of the microbiome on vaccine-induced immunity and viremia control. Our study clearly demonstrates direct effects of a mucosal SIV vaccine regimen on the rectal microbiome and validates our previously reported SIV vaccine-induced sex bias. Sex and the microbiome are critical factors that should not be overlooked in vaccine design and evaluation.IMPORTANCE Differences in HIV pathogenesis between males and females, including immunity postinfection, have been well documented, as have steroid hormone effects on the microbiome, which is known to influence mucosal immune responses. Few studies have applied this knowledge to vaccine trials. We investigated two SIV vaccine regimens combining mucosal priming immunizations and systemic protein boosting. We again report a vaccine-induced sex bias, with female rhesus macaques but not males displaying significantly reduced acute viremia. The vaccine regimens, especially the mucosal primes, significantly altered the rectal microbiome. The greatest effects were in females. Striking differences between female and male macaques in correlations of prevalent rectal bacteria with viral loads and potentially protective immune responses were observed. Effects of the microbiome on vaccine-induced immunity and viremia control require further study by microbiome transfer. However, the findings presented highlight the critical importance of considering effects of sex and the microbiome in vaccine design and evaluation.

Mucosal-associated invariant T (MAIT) cells provide B-cell help in vaccinated and subsequently SIV-infected Rhesus Macaques.

Mucosal-associated invariant T (MAIT) cells help combat opportunistic infections. Thus, MAIT cells are of interest in HIV/SIV vaccination and infection. We investigated MAIT cell dynamics and function in rhesus macaque blood and bronchoalveolar lavage (BAL) following mucosal adenovirus (Ad)-SIV recombinant priming, intramuscular SIV envelope boosting and infection following repeated low-dose intravaginal SIV exposures. Increased frequencies of blood MAIT cells over the course of vaccination were observed, which were maintained even 12-weeks post-SIV infection. BAL MAIT cells only increased after the first Ad immunization. Vaccination increased MAIT cell levels in blood and BAL expressing the antiviral cytokine IFN-γ and TNF-α and the proliferation marker Ki67. Upon T cell-specific α-CD3, α-CD28 stimulation, MAIT cells showed a greater capacity to secrete cytokines/chemokines associated with help for B cell activation, migration and regulation compared to CD3+MR1- cells. Culture of MAIT cell supernatants with B cells led to greater tissue like memory B cell frequencies. MAIT cell frequencies in blood and BAL correlated with SIV-specific antibody levels in rectal secretions and with SIV-specific tissue resident memory B cells. Overall, SIV vaccination influenced MAIT cell frequency and functionality. The potential for MAIT cells to provide help to B cells was evident during both vaccination and infection.

An SAMT-247 Microbicide Provides Potent Protection against Intravaginal Simian Immunodeficiency Virus Infection of Rhesus Macaques, whereas an Added Vaccine Component Elicits Mixed Outcomes.

Because of microbicide noncompliance and lack of a durable, highly effective vaccine, a combined approach might improve HIV prophylaxis. We tested whether a vaccine-microbicide combination would enhance protection against SIV infection in rhesus macaques. Four macaque groups included vaccine only, vaccine-microbicide, microbicide only, and controls. Vaccine groups were primed twice mucosally with replicating adenovirus type 5 host range mutant SIV env/rev, gag, and nef recombinants and boosted twice i.m. with SIV gp120 proteins in alum. Controls and the microbicide-only group received adenovirus type 5 host range mutant empty vector and alum. The microbicide was SAMT-247, a 2-mercaptobenzamide thioester that targets the viral nucleocapsid protein NCp7, causing zinc ejection and preventing RNA encapsidation. Following vaccination, macaques were challenged intravaginally with repeated weekly low doses of SIVmac251 administered 3 h after application of 0.8% SAMT-247 gel (vaccine-microbicide and microbicide groups) or placebo gel (vaccine-only and control groups). The microbicide-only group exhibited potent protection; 10 of 12 macaques remained uninfected following 15 SIV challenges. The vaccine-only group developed strong mucosal and systemic humoral and cellular immunity but did not exhibit delayed acquisition compared with adjuvant controls. However, the vaccine-microbicide group exhibited significant acquisition delay compared with both control and vaccine-only groups, indicating further exploration of the combination strategy is warranted. Impaired protection in the vaccine-microbicide group compared with the microbicide-only group was not attributed to a vaccine-induced increase in SIV target cells. Possible Ab-dependent enhancement will be further investigated. The potent protection provided by SAMT-247 encourages its movement into human clinical trials.

TFH Cells Induced by Vaccination and Following SIV Challenge Support Env-Specific Humoral Immunity in the Rectal-Genital Tract and Circulation of Female Rhesus Macaques.

T follicular helper (TFH) cells are pivotal in lymph node (LN) germinal center (GC) B cell affinity maturation. Circulating CXCR5+ CD4+ T (cTFH) cells have supported memory B cell activation and broadly neutralizing antibodies in HIV controllers. We investigated the contribution of LN SIV-specific TFH and cTFH cells to Env-specific humoral immunity in female rhesus macaques following a mucosal Ad5hr-SIV recombinant priming and SIV gp120 intramuscular boosting vaccine regimen and following SIV vaginal challenge. TFH and B cells were characterized by flow cytometry. B cell help was evaluated in TFH-B cell co-cultures and by real-time PCR. Vaccination induced Env-specific TFH and Env-specific memory (ESM) B cells in LNs. LN Env-specific TFH cells post-priming and GC ESM B cells post-boosting correlated with rectal Env-specific IgA titers, and GC B cells at the same timepoints correlated with vaginal Env-specific IgG titers. Vaccination expanded cTFH cell responses, including CD25+ Env-specific cTFH cells that correlated negatively with vaginal Env-specific IgG titers but positively with rectal Env-specific IgA titers. Although cTFH cells post-2nd boost positively correlated with viral-loads following SIV challenge, cTFH cells of SIV-infected and protected macaques supported maturation of circulating B cells into plasma cells and IgA release in co-culture. Additionally, cTFH cells of naïve macaques promoted upregulation of genes associated with B cell proliferation, BCR engagement, plasma cell maturation, and antibody production, highlighting the role of cTFH cells in blood B cell maturation. Vaccine-induced LN TFH and GC B cells supported anti-viral mucosal immunity while cTFH cells provided B cell help in the periphery during immunization and after SIV challenge. Induction of TFH responses in blood and secondary lymphoid organs is likely desirable for protective efficacy of HIV vaccines.

Dendritic Cells in HIV/SIV Prophylactic and Therapeutic Vaccination.

Dendritic cells (DCs) are involved in human and simian immunodeficiency virus (HIV and SIV) pathogenesis but also play a critical role in orchestrating innate and adaptive vaccine-specific immune responses. Effective HIV/SIV vaccines require strong antigen-specific CD4 T cell responses, cytotoxic activity of CD8 T cells, and neutralizing/non-neutralizing antibody production at mucosal and systemic sites. To develop a protective HIV/SIV vaccine, vaccine regimens including DCs themselves, protein, DNA, mRNA, virus vectors, and various combinations have been evaluated in different animal and human models. Recent studies have shown that DCs enhanced prophylactic HIV/SIV vaccine efficacy by producing pro-inflammatory cytokines, improving T cell responses, and recruiting effector cells to target tissues. DCs are also targets for therapeutic HIV/SIV vaccines due to their ability to reverse latency, present antigen, and augment T and B cell immunity. Here, we review the complex interactions of DCs over the course of HIV/SIV prophylactic and therapeutic immunizations, providing new insights into development of advanced DC-targeted HIV/SIV vaccines.

Differential Effect of Mucosal NKp44+ Innate Lymphoid Cells and Δγ Cells on Simian Immunodeficiency Virus Infection Outcome in Rhesus Macaques.

NK cells are essential for controlling viral infections. We investigated NK cell and innate lymphoid cell (ILC) dynamics and function in rhesus macaque rectal tissue and blood following mucosal priming with replicating adenovirus (Ad)-SIV recombinants, systemic boosting with SIV envelope protein, and subsequent repeated low-dose intravaginal SIV exposures. Mucosal memory-like NK and ILC subsets in rectal and vaginal tissues of chronically infected macaques were also evaluated. No differences in NK cell or ILC frequencies or cytokine production were seen between vaccinated and Ad-empty/alum controls, suggesting responses were due to the Ad-vector and alum vaccine components. Mucosal NKp44+ ILCs increased postvaccination and returned to prelevels postinfection. The vaccine regimen induced mucosal SIV-specific Ab, which mediated Ab-dependent cellular cytotoxicity and was correlated with mucosal NKp44+CD16+ ILCs. Postvaccination NKp44+ and NKp44+IL-17+ ILC frequencies were associated with delayed SIV acquisition and decreased viremia. In chronically SIV-infected animals, NKp44+ ILCs negatively correlated with viral load, further suggesting a protective effect, whereas, NKG2A- NKp44- double-negative ILCs positively correlated with viral load, indicating a pathogenic role. No such associations of circulating NK cells were seen. Δγ NK cells in mucosal tissues of chronically infected animals exhibited impaired cytokine production compared with non-Δγ NK cells but responded to anti-gp120 Ab and Gag peptides, whereas non-Δγ NK cells did not. Mucosal Δγ NKp44+ and Δγ DN cells were similarly associated with protection and disease progression, respectively. Thus, the data suggest NKp44+ ILCs and Δγ cells contribute to SIV infection outcomes. Vaccines that promote mucosal NKp44+ and suppress double-negative ILCs are likely desirable.

Alveolar Macrophage Dysfunction and Increased PD-1 Expression During Chronic SIV Infection of Rhesus Macaques.

HIV infected individuals have been shown to be pre-disposed to pulmonary infections even while receiving anti-retroviral therapy. Alveolar macrophages (AMs) play a critical role in lung innate immunity, but contradictory results have been reported regarding their functionality following HIV infection. Here, using the SIV rhesus macaque model, we document the effect of SIV infection on the phenotypic and functional properties of AMs. Following infection with SIVmac251, AMs in bronchoalveolar lavage (BAL) sampled over 2- to 20-weeks post-infection (wpi) were compared to those in BAL samples from naïve macaques. AM expression of proinflammatory cytokines TNF-α, IL-6, IL-1ß, and chemokine RANTES drastically increased 2-wpi compared to AMs of naïve macaques (p < 0.0001 for all), but dropped significantly with progression to chronic infection. Phagocytic activity of AMs 2-and 4-wpi was elevated compared to AMs of naive animals (p = 0.0005, p = 0.0004, respectively) but significantly decreased by 12-wpi (p = 0.0022, p = 0.0019, respectively). By 20-wpi the ability of AMs from chronically infected animals to perform SIV-specific antibody-dependent phagocytosis (ADP) was also diminished (p = 0.028). Acute SIV infection was associated with increased FcγRIII expression which subsequently declined with disease progression. Frequency of FcγRIII+ AMs showed a strong trend toward correlation with SIV-specific ADP, and at 2-wpi FcγRIII expression negatively correlated with viral load (r = -0.6819; p = 0.0013), suggesting a contribution to viremia control. Importantly, PD-1 was found to be expressed on AMs and showed a strong trend toward correlation with plasma viral load (r = 0.8266; p = 0.058), indicating that similar to over-expression on T-cells, PD-1 expression on AMs may also be associated with disease progression. Further, AMs predominantly expressed PD-L2, which remained consistent over the course of infection. PD-1 blockade enhanced SIV-specific ADP by AMs from chronic infection indicating that the PD-1/PD-L2 pathway may modulate functional activity of AMs at that stage. These findings provide new insight into the dynamics of SIV infection leading to AM dysfunction and alteration of pulmonary innate immunity. Our results suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in HIV-infected individuals.

High-fidelity detection and sorting of nanoscale vesicles in viral disease and cancer.

Biological nanoparticles, including viruses and extracellular vesicles (EVs), are of interest to many fields of medicine as biomarkers and mediators of or treatments for disease. However, exosomes and small viruses fall below the detection limits of conventional flow cytometers due to the overlap of particle-associated scattered light signals with the detection of background instrument noise from diffusely scattered light. To identify, sort, and study distinct subsets of EVs and other nanoparticles, as individual particles, we developed nanoscale Fluorescence Analysis and Cytometric Sorting (nanoFACS) methods to maximise information and material that can be obtained with high speed, high resolution flow cytometers. This nanoFACS method requires analysis of the instrument background noise (herein defined as the "reference noise"). With these methods, we demonstrate detection of tumour cell-derived EVs with specific tumour antigens using both fluorescence and scattered light parameters. We further validated the performance of nanoFACS by sorting two distinct HIV strains to >95% purity and confirmed the viability (infectivity) and molecular specificity (specific cell tropism) of biological nanomaterials sorted with nanoFACS. This nanoFACS method provides a unique way to analyse and sort functional EV- and viral-subsets with preservation of vesicular structure, surface protein specificity and RNA cargo activity.

Knowns and Unknowns of Assaying Antibody-Dependent Cell-Mediated Cytotoxicity Against HIV-1.

It is now well-accepted that Fc-mediated effector functions, including antibody-dependent cellular cytotoxicity (ADCC), can contribute to vaccine-elicited protection as well as post-infection control of HIV viremia. This picture was derived using a wide array of ADCC assays, no two of which are strictly comparable, and none of which is qualified at the clinical laboratory level. An earlier comparative study of assay protocols showed that while data from different ADCC assay formats were often correlated, they remained distinct in terms of target cells and the epitopes and antigen(s) available for recognition by antibodies, the effector cells, and the readout of cytotoxicity. This initial study warrants expanded analyses of the relationships among all current assay formats to determine where they detect overlapping activities and where they do not. Here we summarize knowns and unknowns of assaying ADCC against HIV-1.

A Pathogenic Role for Splenic B1 Cells in SIV Disease Progression in Rhesus Macaques.

B1 cells spontaneously produce protective natural antibodies which provide the first line of defense against a variety of pathogens. Although these natural antibodies share similar autoreactive features with several HIV-1 broadly neutralizing antibodies, the role of B1 cells in HIV/SIV disease progression is unknown. We report the presence of human-like B1 cells in rhesus macaques. During chronic SIV infection, we found that the frequency of splenic CD11b+ B1 cells positively correlated with plasma SIV viral load and exhausted T cells. Mechanistically, we discovered that splenic CD11b+ B1 cells express PD-L2 and IL-10, and were able to induce PD-1 upregulation on CD4+ T cells in vitro. These findings suggest that splenic CD11b+ B1 cells may contribute to the regulation of SIV plasma viral load by enhancing T cell exhaustion. Therefore, understanding the mechanisms that govern their function in rhesus macaques may lead to novel therapeutic strategies for impeding HIV/SIV disease progression.

Replicating Adenovirus-SIV Immunization of Rhesus Macaques Induces Mucosal Dendritic Cell Activation and Function Leading to Rectal Immune Responses.

Inducing strong mucosal immune responses by vaccination is important for providing protection against simian immunodeficiency virus (SIV). A replicating adenovirus type 5 host range mutant vector (Ad5hr) expressing SIV proteins induced mucosal immune responses in rectal tissue associated with delayed SIV acquisition in female rhesus macaques, but the initial mechanisms leading to the induced immunity have not been elucidated. As dendritic cells (DCs) are known to orchestrate both innate and adaptive effector immune cell responses, we investigated their role here. Rhesus macaques were immunized twice mucosally with a replicating Ad5hr expressing SIV Env, Gag, and Nef (Ad-SIV) or empty Ad5hr vector (Ad-Empty). DC subsets and their activation were examined in rectal tissue, blood, and LNs at 3 timepoints after each immunization. Plasmacytoid DCs, myeloid DCs, and Langerhans cells were significantly increased in the rectal mucosa, but only myeloid DCs were significantly increased in blood post-immunizations. All rectal DC subsets showed increased frequencies of cells expressing activation markers and cytokines post-immunization, blood DCs showed mixed results, and LN DCs showed few changes. Rectal DCs responded strongly to the vector rather than expressed SIV antigens, but rectal DC frequencies positively correlated with induced rectal antigen-specific memory T and B cells. These correlations were confirmed by in vitro co-cultures showing that rectal Ad-SIV DCs induced proliferation and antigen-specific cytokine production by autologous naïve T cells. Our results highlight the rapid response of DCs to Ad immunization and their role in mucosal immune activation and identify initial cellular mechanisms of the replicating Ad-SIV vaccine in the rhesus macaque model.

Mucosal vaccine efficacy against intrarectal SHIV is independent of anti-Env antibody response.

It is widely believed that protection against acquisition of HIV or SIV infection requires anti-envelope (anti-Env) antibodies, and that cellular immunity may affect viral loads but not acquisition, except in special cases. Here we provide evidence to the contrary. Mucosal immunization may enhance HIV vaccine efficacy by eliciting protective responses at portals of exposure. Accordingly, we vaccinated macaques mucosally with HIV/SIV peptides, modified vaccinia Ankara-SIV (MVA-SIV), and HIV-gp120-CD4 fusion protein plus adjuvants, which consistently reduced infection risk against heterologous intrarectal SHIVSF162P4 challenge, both high dose and repeated low dose. Surprisingly, vaccinated animals exhibited no anti-gp120 humoral responses above background and Gag- and Env-specific T cells were induced but failed to correlate with viral acquisition. Instead, vaccine-induced gut microbiome alteration and myeloid cell accumulation in colorectal mucosa correlated with protection. Ex vivo stimulation of the myeloid cell-enriched population with SHIV led to enhanced production of trained immunity markers TNF-α and IL-6, as well as viral coreceptor agonist MIP1α, which correlated with reduced viral Gag expression and in vivo viral acquisition. Overall, our results suggest mechanisms involving trained innate mucosal immunity together with antigen-specific T cells, and also indicate that vaccines can have critical effects on the gut microbiome, which in turn can affect resistance to infection. Strategies to elicit similar responses may be considered for vaccine designs to achieve optimal protective efficacy.