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A heterologous Ad26/MVA vaccine was given prior to an analytic treatment interruption (ATI) in people living with HIV-1 (mainly CRF01_AE) who initiated antiretroviral treatment (ART) during acute HIV-1. We investigate the impact of Ad26/MVA vaccination on antibody (Ab)-mediated immune responses and their effect on time to viral rebound. The vaccine mainly triggers vaccine-matched binding Abs while, upon viral rebound post ATI, infection-specific CRF01_AE binding Abs increase in all participants. Binding Abs are not associated with time to viral rebound. The Ad26/MVA mosaic vaccine profile consists of correlated non-CRF01_AE binding Ab and Fc effector features, with strong Ab-dependent cellular phagocytosis (ADCP) responses. CRF01_AE-specific ADCP responses (measured either prior to or post ATI) are significantly higher in individuals with delayed viral rebound. Our results suggest that vaccines eliciting cross-reactive responses with circulating viruses in a target population could be beneficial and that ADCP responses may play a role in viral control post treatment interruption., Competing Interests: Declaration of interests The views expressed are those of the authors and should not be construed to represent the positions of the US Army, the Department of Defense, the Department of Health and Human Services, or the Henry M. Jackson Foundation for the Advancement of Military Medicine. D.J.S., F.L.T., H.S., and M.G.P. were employees of Janssen Vaccines & Prevention at the time the study was conducted and still hold stock in Johnson & Johnson. M.G.P. is an employee of Janssen Vaccines & Prevention and holds stock in Johnson & Johnson. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript., (Copyright © 2024 Henry M Jackson Foundation for the Advancement of Military Medicine, Inc. Published by Elsevier Inc. All rights reserved.)

The repeat emergence of SARS-CoV-2 variants of concern (VoC) with decreased susceptibility to vaccine-elicited antibodies highlights the need to develop next-generation vaccine candidates that confer broad protection. Here we describe the antibody response induced by the SARS-CoV-2 Spike Ferritin Nanoparticle (SpFN) vaccine candidate adjuvanted with the Army Liposomal Formulation including QS21 (ALFQ) in non-human primates. By isolating and characterizing several monoclonal antibodies directed against the Spike Receptor Binding Domain (RBD), N-Terminal Domain (NTD), or the S2 Domain, we define the molecular recognition of vaccine-elicited cross-reactive monoclonal antibodies (mAbs) elicited by SpFN. We identify six neutralizing antibodies with broad sarbecovirus cross-reactivity that recapitulate serum polyclonal antibody responses. In particular, RBD mAb WRAIR-5001 binds to the conserved cryptic region with high affinity to sarbecovirus clades 1 and 2, including Omicron variants, while mAb WRAIR-5021 offers complete protection from B.1.617.2 (Delta) in a murine challenge study. Our data further highlight the ability of SpFN vaccination to stimulate cross-reactive B cells targeting conserved regions of the Spike with activity against SARS CoV-1 and SARS-CoV-2 variants., (© 2024. The Author(s).)

Zika virus (ZIKV) is an emerging pathogen that causes devastating congenital defects. The overlapping epidemiology and immunologic cross-reactivity between ZIKV and dengue virus (DENV) pose complex challenges to vaccine design, given the potential for antibody-dependent enhancement of disease. Therefore, classification of ZIKV-specific antibody targets is of notable value. From a ZIKV-infected rhesus macaque, we identify ZIKV-reactive B cells and isolate potent neutralizing monoclonal antibodies (mAbs) with no cross-reactivity to DENV. We group these mAbs into four distinct antigenic groups targeting ZIKV-specific cross-protomer epitopes on the envelope glycoprotein. Co-crystal structures of representative mAbs in complex with ZIKV envelope glycoprotein reveal envelope-dimer epitope and unique dimer-dimer epitope targeting. All four specificities are serologically identified in convalescent humans following ZIKV infection, and representative mAbs from all four groups protect against ZIKV replication in mice. These results provide key insights into ZIKV-specific antigenicity and have implications for ZIKV vaccine, diagnostic, and therapeutic development., Competing Interests: Declaration of interests S.J.K., V.D., G.D., K.M., N.M., D.H.B., M.G.J., R.S.S., and R.G.J. are named inventors on a PCT patent application WO 2019/209974 describing ZIKV neutralizing antibodies and their use. D.H.B. has received grants from Novavax and personal fees from IGM Biosciences. M.H., A.A., E.D., and B.J.D. are employees of Integral Molecular. B.J.D. is also a shareholder of the company., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Transgender women (TGW) are disproportionally affected by HIV infection, with a global estimated prevalence of 19.9%, often attributed to behavioral risk factors, with less known about biological factors. We evaluated potential biological risk factors for HIV acquisition in TGW at the sites of viral entry by assessing immune parameters of the neovaginal surface and gut mucosa. The neovagina in TGW, compared with the vagina in cisgender women (CW), shows distinct cell composition and may pose a more inflammatory environment, evidenced by increased CD4+ T cell activation and higher levels of soluble markers of inflammation (C-reactive protein, soluble CD30). Increased inflammation may be driven by microbiome composition, as shown by a greater abundance of Prevotella and a higher Shannon Diversity Index. In addition, we have observed higher frequency of CD4+CCR5+ target cells and decreased DNA methylation of the CCR5 gene in the gut mucosa of TGW compared with CW and men who have sex with men, which was inversely correlated with testosterone levels. The rectal microbiome composition in TGW appears to favor a proinflammatory milieu as well as mucosal barrier disruption. Thus, it is possible that increased inflammation and higher frequencies of CCR5-expressing target cells at sites of mucosal viral entry may contribute to increased risk of HIV acquisition in TGW, with further validation in larger studies warranted.

Introduction: Antibody therapeutic strategies have served an important role during the COVID-19 pandemic, even as their effectiveness has waned with the emergence of escape variants. Here we sought to determine the concentration of convalescent immunoglobulin required to protect against disease from SARS-CoV-2 in a Syrian golden hamster model., Methods: Total IgG and IgM were isolated from plasma of SARS-CoV-2 convalescent donors. Dose titrations of IgG and IgM were infused into hamsters 1 day prior to challenge with SARS-CoV-2 Wuhan-1., Results: The IgM preparation was found to have ~25-fold greater neutralization potency than IgG. IgG infusion protected hamsters from disease in a dose-dependent manner, with detectable serum neutralizing titers correlating with protection. Despite a higher in vitro neutralizing potency, IgM failed to protect against disease when transferred into hamsters., Discussion: This study adds to the growing body of literature that demonstrates neutralizing IgG antibodies are important for protection from SARS-CoV-2 disease, and confirms that polyclonal IgG in sera can be an effective preventative strategy if the neutralizing titers are sufficiently high. In the context of new variants, against which existing vaccines or monoclonal antibodies have reduced efficacy, sera from individuals who have recovered from infection with the emerging variant may potentially remain an efficacious tool., Competing Interests: Authors MP, CK, BS, DB and SK are employed by BIOQUAL, Inc. 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 © 2023 King, Dussupt, Mendez-Rivera, Slike, Tran, Jackson, Barkei, Zemil, Tourtellott-Fogt, Kuklis, Soman, Ahmed, Porto, Kitajewski, Spence, Benetiene, Wieczorek, Kar, Gromowski, Polonis, Krebs, Modjarrad and Bolton.)

Introduction: Infants acquire maternal antibodies by Fc receptor transcytosis across the placenta during pregnancy. Fc receptors are expressed on immune cells and are important for activation of effector cell functions., Methods: In this study, we evaluated Fc receptor engagement and ADCC activity of plasma binding antibodies from human immunodeficiency virus-1 (HIV) -infected mothers and to identify factors that may contribute to protection from HIV vertical transmission., Results: HIV-specific binding and Fc receptor engagement of plasma antibodies varied between mothers by transmission status and infants by infection status. Non-transmitting (NT) mothers and HIV-uninfected infants had antibodies with higher neonatal Fc receptor (FcRn) and FcγR engagement, as compared to transmitting (T) mothers and HIV+ infants, respectively. A significant inverse correlation between plasma antibody FcRn and FcγR engagement was observed for T mothers, but not NT mothers. Conversely, a significant direct correlation was observed between plasma antibody FcRn and FcγR engagement for HIV- infants, but not for HIV+ infants. Consequently, we observed significantly higher plasma antibody ADCC potency and breadth in HIV- infants, as compared to HIV+ infants. However, no differences in overall ADCC potency and breadth were observed between mothers. FcRn-engagement of HIV-specific antibodies in both mothers and infants predicted a lack of vertical transmission of HIV., Discussion: This study indicates that HIV-uninfected infants acquire HIV-specific antibodies with greater Fc receptor engagement and thus, greater ADCC capacity., 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., (Copyright © 2022 Barrows, Krebs, Jian, Zemil, Slike, Dussupt, Tran, Mendez-Rivera, Chang, O’Sullivan, Mann, Sanders-Buell, Shubin, Creegan, Paquin-Proulx, Ehrenberg, Laurence-Chenine, Srithanaviboonchai, Thomas, Eller, Ferrari, Robb, Rao, Tovanabutra, Polonis and Wieczorek.)

HIV-1 replication within the central nervous system (CNS) impairs neurocognitive function and has the potential to establish persistent, compartmentalized viral reservoirs. The origins of HIV-1 detected in the CNS compartment are unknown, including whether cells within the cerebrospinal fluid (CSF) produce virus. We measured viral RNA+ cells in CSF from acutely infected macaques longitudinally and people living with early stages of acute HIV-1. Active viral transcription (spliced viral RNA) was present in CSF CD4+ T cells as early as four weeks post-SHIV infection, and among all acute HIV-1 specimens (N = 6; Fiebig III/IV). Replication-inactive CD4+ T cell infection, indicated by unspliced viral RNA in the absence of spliced viral RNA, was even more prevalent, present in CSF of >50% macaques and human CSF at ~10-fold higher frequency than productive infection. Infection levels were similar between CSF and peripheral blood (and lymph nodes in macaques), indicating comparable T cell infection across these compartments. In addition, surface markers of activation were increased on CSF T cells and monocytes and correlated with CSF soluble markers of inflammation. These studies provide direct evidence of HIV-1 replication in CD4+ T cells and broad immune activation in peripheral blood and the CNS during acute infection, likely contributing to early neuroinflammation and reservoir seeding. Thus, early initiation of antiretroviral therapy may not be able to prevent establishment of CNS viral reservoirs and sources of long-term inflammation, important targets for HIV-1 cure and therapeutic strategies., Competing Interests: The authors have declared that no competing interests exist.

Acute HIV-1 infection (AHI) results in the widespread depletion of CD4 + T cells in peripheral blood and gut mucosal tissue. However, the impact on the predominantly CD4 + immunoregulatory invariant natural killer T (iNKT) cells during AHI remains unknown. Here, iNKT cells from peripheral blood and colonic mucosa were investigated during treated and untreated AHI. iNKT cells in blood were activated and rapidly depleted in untreated AHI. At the time of peak HIV-1 viral load, these cells showed the elevated expression of cell death-associated transcripts compared to preinfection. Residual peripheral iNKT cells suffered a diminished responsiveness to in vitro stimulation early into chronic infection. Additionally, HIV-1 DNA, as well as spliced and unspliced viral RNA, were detected in iNKT cells isolated from blood, indicating the active infection of these cells in vivo. The loss of iNKT cells occurred from Fiebig stage III in the colonic mucosa, and these cells were not restored to normal levels after initiation of ART during AHI. CD4 + iNKT cells were depleted faster and more profoundly than conventional CD4 + T cells, and the preferential infection of CD4 + iNKT cells over conventional CD4 + T cells was confirmed by in vitro infection experiments. In vitro data also provided evidence of latent infection in iNKT cells. Strikingly, preinfection levels of peripheral blood CD4 + iNKT cells correlated directly with the peak HIV-1 load. These findings support a model in which iNKT cells are early targets for HIV-1 infection, driving their rapid loss from circulation and colonic mucosa., Competing Interests: Competing interest statement: J.A. previously received honoraria from Merck, ViiV Healthcare, Roche, AbbVie, and Gilead for her participation in advisory meetings., (Copyright © 2021 the Author(s). Published by PNAS.)

Determining which immunological mechanisms contribute to the development of broad neutralizing antibodies (bNAbs) during HIV-1 infection is a major goal to inform vaccine design. Using samples from a longitudinal HIV-1 acute infection cohort, we found key B cell determinants within the first 14-43 days of viremia that predict the development of bNAbs years later. Individuals who develop neutralization breadth had significantly higher B cell engagement with the autologous founder HIV envelope (Env) within 1 month of initial viremia. A higher frequency of founder-Env-specific naive B cells was associated with increased B cell activation and differentiation and predictive of bNAb development. These data demonstrate that the initial B cell interaction with the founder HIV Env is important for the development of broadly neutralizing antibodies and provide evidence that events within HIV acute infection lead to downstream functional outcomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: Activated (CD38HLA-DR) PD-1 CD4 T cells are strongly associated with virus replication and disease progression in untreated HIV-1 infection, and viral persistence in individuals on ART. Few studies have examined cell-associated viral load (CAVL) in different activated CD4 T-cell populations to measure relative contributions to viral reservoirs., Design: Longitudinal assessment of HIV-1 chronically infected Ugandans initiating ART, to investigate activated CD4 T-cell populations and their contribution to viral reservoirs., Methods: We followed 32 HIV-1 chronically infected individuals from Kampala, Uganda, and determined their CD4 T-cell counts and viral load at baseline, 6, and 12 months after the initiation of ART. T-cell populations were sorted based on activation profiles and gag DNA was measured to determine CAVL within these populations. Soluble factors associated with inflammation were measured in plasma using a multiplexed platform., Results: Concomitant with viral load decline and CD4 T-cell count rebound, the activated PD-1 CD4 T-cell population contracted upon initiation of ART. Baseline levels of activated PD-1 CD4 T cells correlated with plasma levels of IP-10 and TNFRII. Interestingly, a higher baseline level of activated PD-1 CD4 T cells was associated with poorer CD4 T-cell recovery after 12 months of ART. This population contributed significantly to the cell-associated HIV DNA load at baseline, whereas their contribution declined on ART, indicating high turnover., Conclusion: Activated PD-1 CD4 T cells are predictors of poor immunologic recovery on ART and may represent a short-lived component of HIV-1 reservoirs.

Mucosa-associated invariant T (MAIT) cell loss in chronic HIV-1 infection is a significant insult to antimicrobial immune defenses. Here we investigate the response of MAIT cells during acute HIV-1 infection utilizing the RV217 cohort with paired longitudinal pre- and post-infection samples. MAIT cells are activated and expand in blood and mucosa coincident with peak HIV-1 viremia, in a manner associated with emerging microbial translocation. This is followed by a phase with elevated function as viral replication is controlled to a set-point level, and later by their functional decline at the onset of chronic infection. Interestingly, enhanced innate-like pathways and characteristics develop progressively in MAIT cells during infection, in parallel with TCR repertoire alterations. These findings delineate the dynamic MAIT cell response to acute HIV-1 infection, and show how the MAIT compartment initially responds and expands with enhanced function, followed by progressive reprogramming away from TCR-dependent antibacterial responses towards innate-like functionality.

Introduction: Liver disease is a common cause of non-AIDS morbidity and mortality in people living with HIV (PLHIV), but the prevalence and significance of liver function test (LFT) abnormalities in early HIV infection is unknown. This study aimed to characterize LFTs in a large cohort of participants with acute HIV infection initiating immediate antiretroviral therapy (ART) and examine the association between LFTs and biomarkers of HIV infection and inflammation., Methods: We measured LFTs at the time of HIV diagnosis and at 4, 12, 24 and 48 weeks after ART initiation in 426 Thai individuals with acute HIV infection from 2009 to 2018. A subset of individuals had data available at 96 and 144 weeks. We excluded individuals with concomitant viral hepatitis. Alanine aminotransferase (ALT) was the primary outcome of interest; values greater than 1.25 times the upper limit of normal were considered elevated. Analyses utilized descriptive statistics, non-parametric tests and multivariate logistic regression., Results: Sixty-six of the 426 individuals (15.5%) had abnormal baseline ALT levels; the majority (43/66, 65.5%) had Grade 1 elevations. Elevated baseline ALT correlated with Fiebig stages III to V (p = 0.001) and baseline HIV RNA >6 log 10 copies/mL (p = 0.012). Baseline elevations resolved by 48 weeks on ART in 59 of the 66 individuals (89%). ALT elevations at 24 and 48 weeks correlated with Fiebig stages I to II at diagnosis (p < 0.001), baseline plasma HIV RNA levels <6 log 10 copies/mL (p < 0.001), abnormal baseline ALT (p < 0.001), baseline CD4 >350 cells/μL (p = 0.03) and older age (p = 0.03). Individuals initiating efavirenz-based regimens were more likely to have elevated ALT levels at 48 weeks compared with those on non-efavirenz-based regimens (p = 0.003)., Conclusions: One in six people with acute HIV infection have elevated LFTs. Clinical outcomes with ART started in acute HIV are generally good, with resolution of ALT elevations within 48 weeks on ART in most cases. These results suggest a multifactorial model for hepatic injury involving a combination of HIV-associated and ART-associated processes, which may change over time., (© 2020 The Authors. Journal of the International AIDS Society published by John Wiley & Sons Ltd on behalf of the International AIDS Society.)

Monocytes play a vital role in HIV-associated neurocognitive disorder (HAND), postulated to transport HIV into the brain and secrete pro-inflammatory cytokines. We analyzed cytokines released by cultured peripheral blood mononuclear cells enriched with the CD14 marker isolated from HIV-infected individuals with HAND and normal cognition (NC) in combination antiretroviral therapy naive and after 1 year on treatment. Interleukin-8 and monocyte chemoattractant protein-1 levels were higher in HAND compared with NC at baseline (P = 0.002 and P < 0.0001). These cytokines remained higher in HAND patients 1 year after combination antiretroviral therapy and were significant when NC patients who were initially HAND were excluded (P = 0.012 and P = 0.002). Both correlated with baseline CD14 peripheral blood mononuclear cell HIV DNA levels supporting the role of HIV DNA reservoir size and monocyte cytokines in HAND persistence.

Modified Vaccinia virus has been shown to be a safe and immunogenic vector platform for delivery of HIV vaccines. Use of this vector is of particular importance to the military, with the implementation of a large scale smallpox vaccination campaign in 2002 in active duty and key civilian personnel in response to potential bioterrorist activities. Humoral immunity to smallpox vaccination was previously shown to be long lasting (up to 75 years) and protective. However, using vaccinia-vectored vaccine delivery for other diseases on a background of anti-vector antibodies (i.e. pre-existing immunity) may limit their use as a vaccine platform, especially in the military. In this pilot study, we examined the durability of vaccinia antibody responses in adult primary vaccinees in a healthy military population using a standard ELISA assay and a novel dendritic cell neutralization assay. We found binding and neutralizing antibody (NAb) responses to vaccinia waned after 5-10 years in a group of 475 active duty military, born after 1972, who were vaccinated as adults with Dryvax®. These responses decreased from a geometric mean titer (GMT) of 250 to baseline (<20) after 10-20 years post vaccination. This contrasted with a comparator group of adults, ages 35-49, who were vaccinated with Dryvax® as children. In the childhood vaccinees, titers persisted for >30 years with a GMT of 210 (range 112-3234). This data suggests limited durability of antibody responses in adult vaccinees compared to those vaccinated in childhood and further that adult vaccinia recipients may benefit similarly from receipt of a vaccinia based vaccine as those who are vaccinia naïve. Our findings may have implications for the smallpox vaccination schedule and support the ongoing development of this promising viral vector in a military vaccination program., Competing Interests: The authors have declared that no competing interests exist.

Objective: To evaluate differences in soluble inflammatory markers between chronically HIV-infected men and women, with or without cognitive impairment, and in response to treatment., Design: Soluble biomarkers were measured in cryopreserved plasma and cerebrospinal fluid (CSF) of 60 treatment-naïve individuals (25 men and 35 women) with chronic HIV infection and 18 HIV-uninfected controls (9 men and 9 women) from Thailand. Following enrollment, participants began combination antiretroviral therapy and were evaluated for expression of these markers after 48 weeks., Methods: Plasma and CSF levels of 19 soluble biomarkers (IFN-γ, TNFα, TNF-RII, IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-15, MCP-1, t-Tau, IP-10, neopterin, IFNα, I-FABP, and sCD14) were measured using either a multiparameter or standard ELISA assay., Results: Prior to combination antiretroviral therapy, women with impaired cognition had elevated levels of neopterin and TNF-RII compared with women with normal cognition in both the plasma and CSF; however, levels did not differ between cognitively impaired or normal men. In a secondary outcome-hypothesis generating analysis, sex differences were also pronounced in plasma levels of MCP-1, IL-10, I-FABP, and sCD14 in response to treatment. Neopterin, IP-10, TNFα, TNF-RII, IFNα, MCP-1, IL-8, I-FABP, and sCD14 plasma levels remained elevated following 48 weeks of therapy in both sexes compared with uninfected controls., Conclusion: We provide evidence of sustained immune activation after 48 weeks of treatment and identify possible sex differences in biomarkers previously linked to cognitive impairment, chronic inflammation, and gut integrity that may contribute to immunological differences between sexes in relationship to disease progression and response to therapy.

Unlabelled: Attrition within the CD4(+)T cell compartment, high viremia, and a cytokine storm characterize the early days after HIV infection. When the first emerging HIV-specific CD8(+)T cell responses gain control over viral replication it is incomplete, and clearance of HIV infection is not achieved even in the rare cases of individuals who spontaneously control viral replication to nearly immeasurably low levels. Thus, despite their partial ability to control viremia, HIV-specific CD8(+)T cell responses are insufficient to clear HIV infection. Studying individuals in the first few days of acute HIV infection, we detected the emergence of a unique population of CD38(+)CD27(-)CD8(+)T cells characterized by the low expression of the CD8 receptor (CD8(dim)). Interestingly, while high frequencies of HIV-specific CD8(+)T cell responses occur within the CD38(+)CD27(-)CD8(dim)T cell population, the minority populations of CD8(bright)T cells are significantly more effective in inhibiting HIV replication. Furthermore, the frequency of CD8(dim)T cells directly correlates with viral load and clinical predictors of more rapid disease progression. We found that a canonical burst of proliferative cytokines coincides with the emergence of CD8(dim)T cells, and the size of this population inversely correlates with the acute loss of CD4(+)T cells. These data indicate, for the first time, that early CD4(+)T cell loss coincides with the expansion of a functionally impaired HIV-specific CD8(dim)T cell population less efficient in controlling HIV viremia., Importance: A distinct population of activated CD8(+)T cells appears during acute HIV infection with diminished capacity to inhibit HIV replication and is predictive of viral set point, offering the first immunologic evidence of CD8(+)T cell dysfunction during acute infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objective: To compare central nervous system (CNS) outcomes in participants treated during acute HIV infection with standard combination antiretroviral therapy (cART) vs. cART plus integrase inhibitor and CCR5 antagonist (cART+)., Design: 24-week randomized open-label prospective evaluation., Method: Participants were evaluated then randomized to initiate cART (efavirenz, tenofovir, and either emtricitabine or lamivudine) vs. cART+ (cART plus raltegravir and maraviroc) during acute HIV and re-evaluated at 4, 12 and 24 weeks. We examined plasma and CSF cytokines, HIV RNA levels, neurological and neuropsychological findings, and brain MRS across groups and compared to healthy controls., Results: At baseline, 62 participants were in Fiebig stages I-V. Randomized groups were similar for mean age (27 vs. 25, p = 0.137), gender (each 94% male), plasma log10 HIV RNA (5.4 vs. 5.6, p = 0.382), CSF log10 HIV RNA (2.35 vs. 3.31, p = 0.561), and estimated duration of HIV (18 vs. 17 days, p = 0.546). Randomized arms did not differ at 24 weeks by any CNS outcome. Combining arms, all measures concurrent with antiretroviral treatment improved, for example, neuropsychological testing (mean NPZ-4 of -0.408 vs. 0.245, p<0.001) and inflammatory markers by MRS (e.g. mean frontal white matter (FWM) choline of 2.92 vs. 2.84, p = 0.045) at baseline and week 24, respectively. Plasma neopterin (p<0.001) and interferon gamma-induced protein 10 (IP-10) (p = 0.007) remained elevated in participants compared to controls but no statistically significant differences were seen in CSF cytokines compared to controls, despite individual variability among the HIV-infected group., Conclusions: A 24-week course of cART+ improved CNS related outcomes, but was not associated with measurable differences compared to standard cART.

Background: The effectiveness of systemic antimonial (sodium stibogluconate, Pentostam, SSG) treatment versus local heat therapy (Thermomed) for cutaneous leishmaniasis was studied previously and showed similar healing rates. We hypothesized that different curative immune responses might develop with systemic and local treatment modalities., Methods: We studied the peripheral blood immune cells in a cohort of 54 cutaneous Leishmania major subjects treated with SSG or TM. Multiparameter flow cytometry, lymphoproliferative assays and cytokine production were analyzed in order to investigate the differences in the immune responses of subjects before, on and after treatment., Results: Healing cutaneous leishmaniasis lead to a significant decline in circulating T cells and NKT-like cells, accompanied by an expansion in NK cells, regardless of treatment modality. Functional changes involved decreased antigen specific CD4+ T cell proliferation (hyporesponsiveness) seen with CD8+ T cell depletion. Moreover, the healing (or healed) state was characterized by fewer circulating regulatory T cells, reduced IFN-γ production and an overall contraction in polyfunctional CD4+ T cells., Conclusion: Healing from cutaneous Leishmaniasis is a dynamic process that alters circulating lymphocyte populations and subsets of T, NK and NKT-like cells. Immunology of healing, through local or systemic treatments, culminated in similar changes in frequency, quality, and antigen specific responsiveness with immunomodulation possibly via a CD8+ T cell dependent mechanism. Understanding the evolving immunologic changes during healing of human leishmaniasis informs protective immune mechanisms.

Objectives: Cognitive impairment remains frequent in HIV, despite combination antiretroviral therapy (cART). Leading theories implicate peripheral monocyte HIV DNA reservoirs as a mechanism for spread of the virus to the brain. These reservoirs remain present despite cART. The objective of this study was to determine if the level of HIV DNA in CD14(+) enriched monocytes predicted cognitive impairment and brain injury., Methods: We enrolled 61 cART-naïve HIV-infected Thais in a prospective study and measured HIV DNA in CD14(+) enriched monocyte samples in a blinded fashion. We determined HAND diagnoses by consensus panel and all participants underwent magnetic resonance spectroscopy (MRS) to measure markers of brain injury. Immune activation was measured via cytokines in cerebrospinal fluid (CSF)., Results: The mean (SD) age was 35 (6.9) years, CD4 T-lymphocyte count was 236 (139) and log10 plasma HIV RNA was 4.8 (0.73). Twenty-eight of 61 met HAND criteria. The log10 CD14(+) HIV DNA was associated with HAND in unadjusted and adjusted models (p = 0.001). There was a 14.5 increased odds ratio for HAND per 1 log-value of HIV DNA (10-fold increase in copy number). Plasma CD14(+) HIV DNA was associated with plasma and CSF neopterin (p = 0.023) and with MRS markers of neuronal injury (lower N-acetyl aspartate) and glial dysfunction (higher myoinositol) in multiple brain regions., Interpretation: Reservoir burden of HIV DNA in monocyte-enriched (CD14(+)) peripheral blood cells increases risk for HAND in treatment-naïve HIV+ subjects and is directly associated with CSF immune activation and both brain injury and glial dysfunction by MRS.

Background: We conducted a novel pilot study comparing different delivery routes of ALVAC-HIV (vCP205), a canarypox vaccine containing HIV gene inserts: env, gag and pol. We explored the concept that direct ex vivo targeting of human dendritic cells (DC) would enhance the immune response compared to either conventional intramuscular or intradermal injections of the vaccine alone., Methodology/principal Findings: Healthy HIV-1 uninfected volunteers were administered ALVAC-HIV or placebo by intramuscular injection (i.m.), intradermal injection (i.d.) or subcutaneous injection (s.q.) of autologous ex vivo transfected DC at months 0, 1, 3 and 6. All vaccine delivery routes were well tolerated. Binding antibodies were observed to both the ALVAC vector and HIV-1 gp160 proteins. Modest cellular responses were observed in 2/7 individuals in the DC arm and 1/8 in the i.m. arm as determined by IFN-γ ELISPOT. Proliferative responses were most frequent in the DC arm where 4/7 individuals had measurable responses to multiple HIV-1 antigens. Loading DC after maturation resulted in lower gene expression, but overall better responses to both HIV-1 and control antigens, and were associated with better IL-2, TNF-α and IFN-γ production., Conclusions/significance: ALVAC-HIV delivered i.m., i.d. or s.q. with autologous ex vivo transfected DC proved to be safe. The DC arm was most immunogenic. Proliferative immune responses were readily detected with only modest cytotoxic CD8 T cell responses. Loading mature DC with the live viral vaccine induced stronger immune responses than loading immature DC, despite increased transgene expression with the latter approach. Volunteers who received the autologous vaccine loaded mature DC developed a broader and durable immune response compared to those vaccinated by conventional routes., Trial Registration: ClinicalTrials.gov NCT00013572.

Aminoacyl tRNA synthetases are known for catalysis of aminoacylation. Significantly, some mammalian synthetases developed cytokine functions possibly linked to disease-causing mutations in tRNA synthetases. Not understood is how epitopes for cytokine signaling were introduced into catalytic scaffolds without disturbing aminoacylation. Here we investigate human tyrosyl-tRNA synthetase, where a catalytic-domain surface helix, next to the active site, was recruited for interleukin-8-like cytokine signaling. Taking advantage of our high resolution structure, the reciprocal impact of rational mutations designed to disrupt aminoacylation or cytokine signaling was investigated with multiple assays. The collective analysis demonstrated a protective fine-structure separation of aminoacylation from cytokine activities within the conserved catalytic domain. As a consequence, disease-causing mutations affecting cell signaling can arise without disturbing aminoacylation. These results with TyrRS also predict the previously unknown binding conformation of interleukin-8-like CXC cytokines.

Dengue viruses (DV), composed of four distinct serotypes (DV1 to DV4), cause 50 to 100 million infections annually. Durable homotypic immunity follows infection but may predispose to severe subsequent heterotypic infections, a risk conferred in part by the immune response itself. Antibody-dependent enhancement (ADE), a process best described in vitro, is epidemiologically linked to complicated DV infections, especially in Southeast Asia. Here we report for the first time the ADE phenomenon in primary human dendritic cells (DC), early targets of DV infection, and human cell lines bearing Fc receptors. We show that ADE is inversely correlated with surface expression of DC-SIGN (DC-specific intercellular adhesion molecule-3-grabbing nonintegrin) and requires Fc gamma receptor IIa (FcgammaRIIa). Mature DC exhibited ADE, whereas immature DC, expressing higher levels of DC-SIGN and similar FcgammaRIIa levels, did not undergo ADE. ADE results in increased intracellular de novo DV protein synthesis, increased viral RNA production and release, and increased infectivity of the supernatants in mature DC. Interestingly, tumor necrosis factor alpha and interleukin-6 (IL-6), but not IL-10 and gamma interferon, were released in the presence of dengue patient sera but generally only at enhancement titers, suggesting a signaling component of ADE. FcgammaRIIa inhibition with monoclonal antibodies abrogated ADE and associated downstream consequences. DV versatility in entry routes (FcgammaRIIa or DC-SIGN) in mature DC broadens target options and suggests additional ways for DC to contribute to the pathogenesis of severe DV infection. Studying the cellular targets of DV infection and their susceptibility to ADE will aid our understanding of complex disease and contribute to the field of vaccine development.

Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases.

Recombinant poxvirus vectors are undergoing intensive evaluation as vaccine candidates for a variety of infectious pathogens. Avipoxviruses, such as canarypox virus, are replication deficient in mammalian cells by virtue of a poorly understood species-specific restriction. Highly attenuated vaccinia virus strains such as modified vaccinia virus Ankara (MVA) are similarly unable to complete replication in most mammalian cells but have an abortive-late phenotype, in that the block to replication occurs post-virus-specific DNA replication. In this study, an identical expression cassette for human immunodeficiency virus gag, pro, and env coding sequences was placed in canarypox virus and MVA vector backbones in order to directly compare vector-borne expression and to analyze differences in vector-host cell interactions. Antigen production by recombinant MVA was shown to be greater than that from recombinant canarypox virus in the mammalian cell lines and in the primary human cells tested. This observation was primarily due to a longer duration of antigen production in recombinant MVA-infected cells. Apoptosis induction was found to be more profound with the empty canarypox virus vector than with MVA. Remarkably, however, the inclusion of a gag/pro/env expression cassette altered the kinetics of apoptosis induction in recombinant MVA-infected cells to levels equal to those found in canarypox virus-infected cells. Antigen production by MVA was noted to be greater in human dendritic cells and resulted in enhanced T-cell stimulation in an in vitro antigen presentation assay. These results reveal differences in poxvirus vector-host cell interactions that should be relevant to their use as immunization vehicles.

The first step of protein synthesis is catalyzed by aminoacyl-tRNA synthetases. In addition, certain mammalian tRNA synthetases link protein synthesis to cytokine signaling pathways. In particular, human tyrosyl-tRNA synthetase (TyrRS) can be split by proteolysis into two fragments having distinct cytokine activities. One of the TyrRS fragments (mini TyrRS) contains features identical to those in CXC chemokines (like interleukin-8) that also act as angiogenic factors. Here mini TyrRS (but not full-length TyrRS) is shown to stimulate chemotaxis of endothelial cells in vitro and stimulate angiogenesis in each of two in vivo animal models. The angiogenic activity of mini TyrRS can be opposed by anti-angiogenic chemokines like IP-10. Thus, a biological fragment of human tyrosyl-tRNA synthetase links protein synthesis to regulation of angiogenesis.

Aminoacyl-tRNA synthetases catalyze the first step of protein synthesis. It was shown recently that human tyrosyl-tRNA synthetase (TyrRS) can be split into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. Tryptophanyl-tRNA synthetase (TrpRS) is a close homologue of TyrRS. A natural fragment, herein designated as mini TrpRS, was shown by others to be produced by alternative splicing. Production of this fragment is reported to be stimulated by IFN-gamma, a cytokine that also stimulates production of angiostatic factors. Mini TrpRS is shown here to be angiostatic in a mammalian cell culture system, the chicken embryo, and two independent angiogenesis assays in the mouse. The full-length enzyme is inactive in the same assays. Thus, protein synthesis may be linked to the regulation of angiogenesis by a natural fragment of TrpRS.

Pathological angiogenesis contributes directly to profound loss of vision associated with many diseases of the eye. Recent work suggests that human tyrosyl- and tryptophanyl-tRNA synthetases (TrpRS) link protein synthesis to signal transduction pathways including angiogenesis. In this study, we show that a recombinant form of a COOH-terminal fragment of TrpRS is a potent antagonist of vascular endothelial growth factor-induced angiogenesis in a mouse model and of naturally occurring retinal angiogenesis in the neonatal mouse. The angiostatic activity is dose-dependent in both systems. The recombinant fragment is similar in size to one generated naturally by alternative splicing and can be produced by proteolysis of the full-length protein. In contrast, the full-length protein is inactive as an antagonist of angiogenesis. These results suggest that fragments of TrpRS, as naturally occurring and potentially nonimmunogenic anti-angiogenics, can be used for the treatment of neovascular eye diseases.