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A major obstacle to achieving long-term antiretroviral (ART) free remission or functional cure of HIV infection is the presence of persistently infected cells that establish a long-lived viral reservoir. HIV largely resides in anatomical regions that are inaccessible to routine sampling, however, and non-invasive methods to understand the longitudinal tissue-wide burden of HIV persistence are urgently needed. Positron emission tomography (PET) imaging is a promising strategy to identify and characterize the tissue-wide burden of HIV. Here, we assess the efficacy of using immunoPET imaging to characterize HIV reservoirs and identify anatomical foci of persistent viral transcriptional activity using a radiolabeled HIV Env-specific broadly neutralizing antibody, 89Zr-VRC01, in HIV-infected individuals with detectable viremia and on suppressive ART compared to uninfected controls (NCT03729752). We also assess the relationship between PET tracer uptake in tissues and timing of ART initiation and direct HIV protein expression in CD4 T cells obtained from lymph node biopsies. We observe significant increases in 89Zr-VRC01 uptake in various tissues (including lymph nodes and gut) in HIV-infected individuals with detectable viremia (N = 5) and on suppressive ART (N = 5) compared to uninfected controls (N = 5). Importantly, PET tracer uptake in inguinal lymph nodes in viremic and ART-suppressed participants significantly and positively correlates with HIV protein expression measured directly in tissue. Our strategy may allow non-invasive longitudinal characterization of residual HIV infection and lays the framework for the development of immunoPET imaging in a variety of other infectious diseases.

Although many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus-specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and nonhuman primates naturally controlling HIV/SIV infection express more of the transcription factor TCF-1 than noncontrollers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and expansion capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 contributes to the regulation of the stem-like memory property of secondary expansion capacity of HIV-specific CD8+ T cells, and they provide a rationale for exploring the enhancement of this pathway in T cell-based therapeutic strategies for HIV.

Immunization with recombinant ALVAC/gp120 alum vaccine provided modest protection from human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) acquisition in humans and macaques. Vaccine-mediated protection was associated with the elicitation of IgG against the envelope V2 loop and of envelope-specific CD4+ T cell responses. We hypothesized that the simultaneous expression of the costimulatory molecule CD40L (CD154) by the ALVAC-HIV vector could increase both protective humoral and cellular responses. We engineered an ALVAC-SIV coexpressing CD40L with SIVmac251 (ALVAC-SIV/CD40L) gag, pol, and env genes. We compared its immunogenicity in macaques with that of a canonical ALVAC-SIV, with both given as a vector-prime/gp120 in alum boost strategy. The ALVAC-SIV/CD40L was superior to the ALVAC-SIV regimen in inducing binding and tier 1 neutralizing antibodies against the gp120. The increase in humoral responses was associated with the expression of the membrane-bound form of the CD40L by CD4+ T cells in lymph nodes. Unexpectedly, the ALVAC-SIV/CD40L vector had a blunting effect on CD4+ Th1 helper responses and instead favored the induction of myeloid-derived suppressor cells, the immune-suppressive interleukin-10 (IL-10) cytokine, and the down-modulatory tryptophan catabolism. Ultimately, this strategy failed to protect macaques from SIV acquisition. Taken together, these results underlie the importance of balanced vaccine-induced activating versus suppressive immune responses in affording protection from HIV.IMPORTANCE CD40-CD40 ligand (CD40L) interaction is crucial for inducing effective cytotoxic and humoral responses against pathogens. Because of its immunomodulatory function, CD40L has been used to enhance immune responses to vaccines, including candidate vaccines for HIV. The only successful vaccine ever tested in humans utilized a strategy combining canarypox virus-based vector (ALVAC) together with an envelope protein (gp120) adjuvanted in alum. This strategy showed limited efficacy in preventing HIV-1/SIV acquisition in humans and macaques. In both species, protection was associated with vaccine-induced antibodies against the HIV envelope and CD4+ T cell responses, including type 1 antiviral responses. In this study, we tested whether augmenting CD40L expression by coexpressing it with the ALVAC vector could increase the protective immune responses. Although coexpression of CD40L did increase humoral responses, it blunted type 1 CD4+ T cell responses against the SIV envelope protein and failed to protect macaques from viral infection.

Qualitative differences in the innate and adaptive responses elicited by different HIV vaccine candidates have not been thoroughly investigated. We tested the ability of the Aventis Pasteur live recombinant canarypox vector (ALVAC)-SIV, DNA-SIV and Ad26-SIV vaccine prime modalities together with two ALVAC-SIV + gp120 protein boosts to reduce the risk of SIVmac251 acquisition in rhesus macaques. We found that the DNA and ALVAC prime regimens were effective, but the Ad26 prime was not. The activation of hypoxia and the inflammasome in CD14+CD16- monocytes, gut-homing CCR5-negative CD4+ T helper 2 (TH2) cells and antibodies to variable region 2 correlated with a decreased risk of SIVmac251 acquisition. By contrast, signal transducer and activator of transcription 3 activation in CD16+ monocytes was associated with an increased risk of virus acquisition. The Ad26 prime regimen induced the accumulation of CX3CR1+CD163+ macrophages in lymph nodes and of long-lasting CD4+ TH17 cells in the gut and lungs. Our data indicate that the selective engagement of monocyte subsets following a vaccine prime influences long-term immunity, uncovering an unexpected association of CD14+ innate monocytes with a reduced risk of SIVmac251 acquisition.

Targeted HIV cure strategies require definition of the mechanisms that maintain the virus. Here, we tracked HIV replication and the persistence of infected CD4 T cells in individuals with natural virologic control by sequencing viruses, T cell receptor genes, HIV integration sites, and cellular transcriptomes. Our results revealed three mechanisms of HIV persistence operating within distinct anatomic and functional compartments. In lymph node, we detected viruses with genetic and transcriptional attributes of active replication in both T follicular helper (TFH) cells and non-TFH memory cells. In blood, we detected inducible proviruses of archival origin among highly differentiated, clonally expanded cells. Linking the lymph node and blood was a small population of circulating cells harboring inducible proviruses of recent origin. Thus, HIV replication in lymphoid tissue, clonal expansion of infected cells, and recirculation of recently infected cells act together to maintain the virus in HIV controllers despite effective antiviral immunity.

A recombinant vaccine containing Aventis Pasteur's canarypox vector (ALVAC)-HIV and gp120 alum decreased the risk of HIV acquisition in the RV144 vaccine trial. The substitution of alum with the more immunogenic MF59 adjuvant is under consideration for the next efficacy human trial. We found here that an ALVAC-simian immunodeficiency virus (SIV) and gp120 alum (ALVAC-SIV + gp120) equivalent vaccine, but not an ALVAC-SIV + gp120 MF59 vaccine, was efficacious in delaying the onset of SIVmac251 in rhesus macaques, despite the higher immunogenicity of the latter adjuvant. Vaccine efficacy was associated with alum-induced, but not with MF59-induced, envelope (Env)-dependent mucosal innate lymphoid cells (ILCs) that produce interleukin (IL)-17, as well as with mucosal IgG to the gp120 variable region 2 (V2) and the expression of 12 genes, ten of which are part of the RAS pathway. The association between RAS activation and vaccine efficacy was also observed in an independent efficacious SIV-vaccine approach. Whether RAS activation, mucosal ILCs and antibodies to V2 are also important hallmarks of HIV-vaccine efficacy in humans will require further studies.

BackgroundLittle is known about the expression of inhibitory molecules cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed-death-1 (PD-1) on Mycobacterium tuberculosis (Mtb)-specific CD4 T-cells and how their expression is impacted by TB treatment.MethodsCryopreserved PBMCs from HIV-TB co-infected and TB mono-infected patients with untreated and treated tuberculosis (TB) disease were stimulated for six hours with PPD and stained. Using polychromatic flow cytometry, we characterized the differentiation state, cytokine profile, and inhibitory molecule expression on PPD-specific CD4 T-cells.ResultsIn our HIV-TB co-infected cohort, TB treatment increased the proportion of PPD-specific CD4 T-cells co-producing IFN-γ+IL-2+TNF-α+ and IFN-γ+IL-2+ (p = 0.0004 and p = 0.0002, respectively) while decreasing the proportion of PPD-specific CD4 T-cells co-producing IFN-γ+MIP1-β+TNF-α+ and IFN-γ+MIP1-β+. The proportion of PPD-specific CD4 T-cells expressing an effector memory phenotype decreased (63.6% vs 51.6%, p = 0.0015) while the proportion expressing a central memory phenotype increased (7.8% vs. 21.7%, p = 0.001) following TB treatment. TB treatment reduced the proportion of PPD-specific CD4 T-cells expressing CTLA-4 (72.4% vs. 44.3%, p = 0.0005) and PD-1 (34.5% vs. 29.2%, p = 0.03). Similar trends were noted in our TB mono-infected cohort.ConclusionTB treatment alters the functional profile of Mtb-specific CD4 T-cells reflecting shifts towards a less differentiated maturational profile and decreases PD-1 and CTLA-4 expression. These could serve as markers of reduced mycobacterial burden. Further study is warranted.

Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5) boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART). Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB) and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC). We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir.

BackgroundAchievement of a cure for HIV infection might need reactivation of latent virus and improvement of HIV-specific immunity. As an initial step, in this trial we assessed the effect of antiretroviral therapy intensification and immune modulation with a DNA prime and recombinant adenovirus 5 (rAd5) boost vaccine.MethodsIn this multicentre, randomised, open-label, non-comparative, phase 2 clinical trial, we enrolled eligible adults 18-70 years of age with chronic HIV-1 infection on suppressive antiretroviral therapy with current CD4 count of at least 350 cells per μL and HIV DNA between 10 and 1000 copies per 10(6) peripheral blood mononuclear cells. After an 8 week lead-in of antiretroviral intensification therapy (standard dose raltegravir and dose-adjusted maraviroc based on baseline antiretroviral therapy), patients were randomly assigned (1:1) to receive antiretroviral therapy intensification alone or intensification plus injections of HIV DNA prime vaccine (4 mg VRC-HIVDNA016-00-VP) at weeks 8, 12, and 16, followed by HIV rAd5 boost vaccine (10(10) particle units of VRC-HIVADV014-00-VP) at week 32. Randomisation was computer generated in permuted blocks of six and was stratified by study site. The primary endpoint was a 0·5 log10 or greater decrease in HIV DNA in peripheral blood mononuclear cells at week 56. This study is registered with ClinicalTrials.gov, number NCT00976404.FindingsBetween Nov 29, 2010, and Oct 28, 2011, we enrolled 28 eligible patients from three academic HIV clinics in the USA. After the 8 week lead-in of antiretroviral intensification therapy, 14 patients were randomly assigned to continue antiretroviral therapy intensification alone and 14 to intensification plus vaccine. Enrolled participants had median CD4 count of 636 cells per μL, median HIV DNA 170 copies per 10(6) peripheral blood mononuclear cells, and duration of antiretroviral therapy of 13 years. The median amount of HIV DNA did not change significantly between baseline and week 56 in the antiretroviral therapy intensification plus vaccine group. One participant in the antiretroviral therapy intensification alone group reached the primary endpoint, with 0·55 log10 decrease in HIV DNA in peripheral blood mononuclear cells. Both treatments were well tolerated. No severe or systemic reactions to vaccination occurred, and five serious adverse events were recorded during the study, most of which resolved spontaneously or were judged unrelated to study treatments.InterpretationAntiretroviral therapy intensification followed by DNA prime and rAd5 boost vaccine did not significantly increase HIV expression or reduce the latent HIV reservoir. A multifaceted approach that includes stronger activators of HIV expression and novel immune modulators will probably be needed to reduce the latent HIV reservoir and allow for long-term control in patients off antiretroviral therapy.FundingObjectif Recherche Vaccin SIDA (ORVACS).

Background: Seasonal influenza results in significant morbidity and mortality worldwide, but the currently licensed inactivated vaccines generally have low vaccine efficacies and could be improved. In this phase 1 clinical trial, we compared seasonal influenza vaccine regimens with different priming strategies, prime-boost intervals, and administration routes to determine the impact of these variables on the resulting antibody response. Methods: Between August 17, 2012 and January 25, 2013, four sites enrolled healthy adults 18–70 years of age. Subjects were randomized to receive one of the following vaccination regimens: trivalent hemagglutinin (HA) DNA prime followed by trivalent inactivated influenza vaccine (IIV3) boost with a 3.5 month interval (DNA-IIV3), IIV3 prime followed by IIV3 boost with a 10 month interval (IIV3-IIV3), or concurrent DNA and IIV3 prime followed by IIV3 boost with a 10 month interval (DNA/IIV3-IIV3). Each regimen was additionally stratified by an IIV3 administration route of either intramuscular (IM) or intradermal (ID). DNA vaccines were administered by a needle-free jet injector (Biojector). Study objectives included evaluating the safety and tolerability of each regimen and measuring the antibody response by hemagglutination inhibition (HAI). Results: Three hundred and sixteen subjects enrolled. Local reactogenicity was mild to moderate in severity, with higher frequencies recorded following DNA vaccine administered by Biojector compared to IIV3 by either route (p <0.02 for pain, swelling, and redness) and following IIV3 by ID route compared to IM route (p <0.001 for swelling and redness). Systemic reactogenicity was similar between regimens. Though no overall differences were observed between regimens, the highest titers post boost were observed in the DNA-IIV3 group by ID route and in the IIV3-IIV3 group by IM route. Conclusions: All vaccination regimens were found to be safe and tolerable. While there were no overall differences between regimens, the DNA-IIV3 group by ID route, and the IIV3-IIV3 group by IM route, showed higher responses compared to the other same-route regimens. [ABSTRACT FROM AUTHOR]

Chimeric Simian-Human Immunodeficiency Viruses (SHIVs) are an important tool for evaluating anti-HIV Env interventions in nonhuman primate (NHP) models. However, most unadapted SHIVs do not replicate well in vivo limiting their utility. Furthermore, adaptation in vivo often negatively impacts fundamental properties of the Env, including neutralization profiles. Transmitted/founder (T/F) viruses are particularly important to study since they represent viruses that initiated primary HIV-1 infections and may have unique attributes. Here we combined in vivo competition and rational design to develop novel subtype C SHIVs containing T/F envelopes. We successfully generated 19 new, infectious subtype C SHIVs, which were tested in multiple combinatorial pools in Indian-origin rhesus macaques. Infected animals attained peak viremia within 5 weeks ranging from 103 to 107 vRNA copies/mL. Sequence analysis during primary infection revealed 7 different SHIVs replicating in 8 productively infected animals with certain clones prominent in each animal. We then generated 5 variants each of 6 SHIV clones (3 that predominated and 3 undetectable after pooled in vivo inoculations), converting a serine at Env375 to methionine, tyrosine, histidine, tryptophan or phenylalanine. Overall, most Env375 mutants replicated better in vitro and in vivo than wild type with both higher and earlier peak viremia. In 4 of these SHIV clones (with and without Env375 mutations) we also created mutations at position 281 to include serine, alanine, valine, or threonine. Some Env281 mutations imparted in vitro replication dynamics similar to mutations at 375; however, clones with both mutations did not exhibit incremental benefit. Therefore, we identified unique subtype C T/F SHIVs that replicate in rhesus macaques with improved acute phase replication kinetics without altering phenotype. In vivo competition and rational design can produce functional SHIVs with globally relevant HIV-1 Envs to add to the growing number of SHIV clones for HIV-1 research in NHPs. [ABSTRACT FROM AUTHOR]

Background: Children are susceptible to severe influenza infections and facilitate community transmission. One potential strategy to improve vaccine immunogenicity in children against seasonal influenza involves a trivalent hemagglutinin DNA prime-trivalent inactivated influenza vaccine (IIV3) boost regimen. Methods: Sites enrolled adolescents, followed by younger children, to receive DNA prime (1 mg or 4 mg) intramuscularly by needle-free jet injector (Biojector), followed by split virus 2012/13 seasonal IIV3 boost by needle and syringe approximately 18 weeks later. A comparator group received IIV3 prime and boost at similar intervals. Primary study objectives included evaluation of the safety and tolerability of the vaccine regimens, with secondary objectives of measuring antibody responses at four weeks post boost by hemagglutination inhibition (HAI) and neutralization assays. Results: Seventy-five children ≥6 to ≤17 years old enrolled. Local reactogenicity was higher after DNA prime compared to IIV3 prime (p<0.001 for pain/tenderness, redness, or swelling), but symptoms were mild to moderate in severity. Systemic reactogenicity was similar between vaccines. Overall, antibody responses were similar among groups, although HAI antibodies revealed a trend towards higher responses following 4 mg DNA-IIV3 compared to IIV3-IIV3. The fold increase of HAI antibodies to A/California/07/2009 [A(H1N1)pdm09] was significantly greater following 4 mg DNA-IIV3 (10.12 fold, 5.60–18.27 95%CI) compared to IIV3-IIV3 (3.86 fold, 2.32–6.44 95%CI). Similar neutralizing titers were observed between regimens, with a trend towards increased response frequencies in 4 mg DNA-IIV3. However, significant differences in fold increase, reported as geometric mean fold ratios, were detected against the H1N1 viruses within the neutralization panel: A/New Caledonia/20/1999 (1.41 fold, 1.10–1.81 95%CI) and A/South Carolina/1/1918 (1.55 fold, 1.27–1.89 95%CI). Conclusions: In this first pediatric DNA vaccine study conducted in the U.S., the DNA prime-IIV3 boost regimen was safe and well tolerated. In children, the 4 mg DNA-IIV3 regimen resulted in antibody responses comparable to the IIV3-IIV3 regimen. [ABSTRACT FROM AUTHOR]

Fatal outcomes of Ebola virus (EBOV) infections are typically preceded by a ‘sepsis-like’ syndrome and lymphopenia despite T cells being resistant to Ebola infection. The mechanisms that lead to T lymphocytes death remain largely unknown; however, the degree of lymphopenia is highly correlative with fatalities. Here we investigated whether the addition of EBOV or its envelope glycoprotein (GP) to isolated primary human CD4+ T cells induced cell death. We observed a significant decrease in cell viability in a GP-dependent manner, which is suggestive of a direct role of GP in T cell death. Using immunoprecipitation assays and flow cytometry, we demonstrate that EBOV directly binds to CD4+ T cells through interaction of GP with TLR4. Transcriptome analysis revealed that the addition of EBOV to CD4+ T cells results in the significant upregulation of pathways associated with interferon signaling, pattern recognition receptors and intracellular activation of NFκB signaling pathway. Both transcriptome analysis and specific inhibitors allowed identification of apoptosis and necrosis as mechanisms associated with the observed T cell death following exposure to EBOV. The addition of the TLR4 inhibitor CLI-095 significantly reduced CD4+ T cell death induced by GP. EBOV stimulation of primary CD4+ T cells resulted in a significant increase in secreted TNFα; inhibition of TNFα-mediated signaling events significantly reduced T cell death while inhibitors of both necrosis and apoptosis similarly reduced EBOV-induced T cell death. Lastly, we show that stimulation with EBOV or GP augments monocyte maturation as determined by an overall increase in expression levels of markers of differentiation. Subsequently, the increased rates of cellular differentiation resulted in higher rates of infection further contributing to T cell death. These results demonstrate that GP directly subverts the host’s immune response by increasing the susceptibility of monocytes to EBOV infection and triggering lymphopenia through direct and indirect mechanisms. [ABSTRACT FROM AUTHOR]

Background: Neutralizing antibodies develop in natural HIV-1 infection. Their development often takes several years and may rely on chronic virus exposure. At the same time recent studies show that treatment early in infection may provide opportunities for immune preservation. However, it is unknown how intermittent treatment in early infection affects development of the humoral immune response over time. We investigate the effect of cART in early HIV infection on the properties of the memory B cell compartment following 6 months of cART or in the absence of treatment. The patients included participated in the Primo-SHM trial where patients with an early HIV-1 infection were randomized to no treatment or treatment for 24 or 60 weeks. Methods: Primo-SHM trial patients selected for the present study were untreated (n = 23) or treated for 24 weeks (n = 24). Here we investigate memory B cell properties at viral set-point and at a late time point (respectively median 54 and 73 weeks) before (re)-initiation of treatment. Results: At viral set-point, the memory B cell compartment in treated patients demonstrated significantly lower fractions of antigen-primed, activated, memory B cells (p = 0.006). In contrast to untreated patients, in treated patients the humoral HIV-specific response reached a set point over time. At a transcriptional level, sets of genes that showed enhanced expression in memory B cells at viral setpoint in untreated patients, conversely showed rapid increase of expression of the same genes in treated patients at the late time point. Conclusion: These data suggest that, although the memory B cell compartment is phenotypically preserved until viral setpoint after treatment interruption, the development of the HIV-specific antibody response may benefit from exposure to HIV. The effect of viral exposure on B cell properties is also reflected by longitudinal changes in transcriptional profile in memory B cells over time in early treated patients. [ABSTRACT FROM AUTHOR]

Ebola virus (EBOV) infections are characterized by deficient T-lymphocyte responses, T-lymphocyte apoptosis and lymphopenia. We previously showed that disabling of interferon-inhibiting domains (IIDs) in the VP24 and VP35 proteins effectively unblocks maturation of dendritic cells (DCs) and increases the secretion of cytokines and chemokines. Here, we investigated the role of IIDs in adaptive and innate cell-mediated responses using recombinant viruses carrying point mutations, which disabled IIDs in VP24 (EBOV/VP24m), VP35 (EBOV/VP35m) or both (EBOV/VP35m/VP24m). Peripheral blood mononuclear cells (PBMCs) from cytomegalovirus (CMV)-seropositive donors were inoculated with the panel of viruses and stimulated with CMV pp65 peptides. Disabling of the VP35 IID resulted in increased proliferation and higher percentages of CD4+ T cells secreting IFNγ and/or TNFα. To address the role of aberrant DC maturation in the IID-mediated suppression of T cell responses, CMV-stimulated DCs were infected with the panel of viruses and co-cultured with autologous T-lymphocytes. Infection with EBOV/VP35m infection resulted in a significant increase, as compared to wt EBOV, in proliferating CD4+ cells secreting IFNγ, TNFα and IL-2. Experiments with expanded CMV-specific T cells demonstrated their increased activation following co-cultivation with CMV-pulsed DCs pre-infected with EBOV/VP24m, EBOV/VP35m and EBOV/VP35m/VP24m, as compared to wt EBOV. Both IIDs were found to block phosphorylation of TCR complex-associated adaptors and downstream signaling molecules. Next, we examined the effects of IIDs on the function of B cells in infected PBMC. Infection with EBOV/VP35m and EBOV/VP35m/VP24m resulted in significant increases in the percentages of phenotypically distinct B-cell subsets and plasma cells, as compared to wt EBOV, suggesting inhibition of B cell function and differentiation by VP35 IID. Finally, infection with EBOV/VP35m increased activation of NK cells, as compared to wt EBOV. These results demonstrate a global suppression of cell-mediated responses by EBOV IIDs and identify the role of DCs in suppression of T-cell responses. [ABSTRACT FROM AUTHOR]

Background: VRC 012 was a Phase I study of a prototype recombinant adenoviral-vector serotype-35 (rAd35) HIV vaccine, the precursor to two recently published clinical trials, HVTN 077 and 083. On the basis of prior evaluation of multiclade rAd5 HIV vaccines, Envelope A (EnvA) was selected as the standard antigen for a series of prototype HIV vaccines to compare various vaccine platforms. In addition, prior studies of rAd5-vectored vaccines suggested pre-existing human immunity may be a confounding factor in vaccine efficacy. rAd35 is less seroprevalent across human populations and was chosen for testing alone and in combination with a rAd5-EnvA vaccine in the present two-part phase I study. Methods: First, five subjects each received a single injection of 109, 1010, or 1011 particle units (PU) of rAd35-EnvA in an open-label, dose-escalation study. Next, 20 Ad5/Ad35-seronegative subjects were randomized to blinded, heterologous prime-boost schedules combining rAd5-EnvA and rAd35-EnvA with a three month interval. rAd35-EnvA was given at 1010 or 1011 PU to ten subjects each; all rAd5-EnvA injections were 1010 PU. EnvA-specific immunogenicity was assessed four weeks post-injection. Solicited reactogenicity and clinical safety were followed after each injection. Results: Vaccinations were well tolerated at all dosages. Antibody responses measured by ELISA were detected at 4 weeks in 30% and 50% of subjects after single doses of 1010 or 1011 PU rAd35, respectively, and in 89% after a single rAd5-EnvA 1010 PU injection. EnvA-specific IFN-γ ELISpot responses were detected at four weeks in 0%, 70%, and 50% of subjects after the respective rAd35-EnvA dosages compared to 89% of subjects after rAd5. T cell responses were higher after a single rAd5-EnvA 1010 PU injection than after a single rAd35-EnvA 1010 PU injection, and humoral responses were low after a single dose of either vector. Of those completing the vaccine schedule, 100% of rAd5-EnvA recipients and 90% of rAd35-EnvA recipients had both T cell and humoral responses after boosting with the heterologous vector. ELISpot response magnitude was similar in both regimens and comparable to a single dose of rAd5. A trend toward more robust CD8 T cell responses using rAd5-EnvA prime and rAd35-EnvA boost was observed. Humoral response magnitude was also similar after either heterologous regimen, but was several fold higher than after a single dose of rAd5. Adverse events (AEs) related to study vaccines were in general mild and limited to one episode of hematuria, Grade two. Activated partial thromboplastin time (aPTT) AEs were consistent with an in vitro effect on the laboratory assay for aPTT due to a transient induction of anti-phospholipid antibody, a phenomenon that has been reported in other adenoviral vector vaccine trials. Conclusions: Limitations of the rAd vaccine vectors, including the complex interactions among pre-existing adenoviral immunity and vaccine-induced immune responses, have prompted investigators to include less seroprevalent vectors such as rAd35-EnvA in prime-boost regimens. The rAd35-EnvA vaccine described here was well tolerated and immunogenic. While it effectively primed and boosted antibody responses when given in a reciprocal prime-boost regimen with rAd5-EnvA using a three-month interval, it did not significantly improve the frequency or magnitude of T cell responses above a single dose of rAd5. The humoral and cellular immunogenicity data reported here may inform future vaccine and study design. Trial Registration: ClinicalTrials.gov [ABSTRACT FROM AUTHOR]

Background: Little is known about the expression of inhibitory molecules cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed-death-1 (PD-1) on Mycobacterium tuberculosis (Mtb)-specific CD4 T-cells and how their expression is impacted by TB treatment. Methods: Cryopreserved PBMCs from HIV-TB co-infected and TB mono-infected patients with untreated and treated tuberculosis (TB) disease were stimulated for six hours with PPD and stained. Using polychromatic flow cytometry, we characterized the differentiation state, cytokine profile, and inhibitory molecule expression on PPD-specific CD4 T-cells. Results: In our HIV-TB co-infected cohort, TB treatment increased the proportion of PPD-specific CD4 T-cells co-producing IFN-γ+IL-2+TNF-α+ and IFN-γ+IL-2+ (p = 0.0004 and p = 0.0002, respectively) while decreasing the proportion of PPD-specific CD4 T-cells co-producing IFN-γ+MIP1-β+TNF-α+ and IFN-γ+MIP1-β+. The proportion of PPD-specific CD4 T-cells expressing an effector memory phenotype decreased (63.6% vs 51.6%, p = 0.0015) while the proportion expressing a central memory phenotype increased (7.8% vs. 21.7%, p = 0.001) following TB treatment. TB treatment reduced the proportion of PPD-specific CD4 T-cells expressing CTLA-4 (72.4% vs. 44.3%, p = 0.0005) and PD-1 (34.5% vs. 29.2%, p = 0.03). Similar trends were noted in our TB mono-infected cohort. Conclusion: TB treatment alters the functional profile of Mtb-specific CD4 T-cells reflecting shifts towards a less differentiated maturational profile and decreases PD-1 and CTLA-4 expression. These could serve as markers of reduced mycobacterial burden. Further study is warranted. [ABSTRACT FROM AUTHOR]

Background: Needle-free delivery improves the immunogenicity of DNA vaccines but is also associated with more local reactogenicity. Here we report the first comparison of Biojector and needle administration of a candidate rAd5 HIV vaccine. Methods: Thirty-one adults, 18–55 years, 20 naive and 11 prior rAd5 vaccine recipients were randomized to receive single rAd5 vaccine via needle or Biojector IM injection at 1010 PU in a Phase I open label clinical trial. Solicited reactogenicity was collected for 5 days; clinical safety and immunogenicity follow-up was continued for 24 weeks. Results: Overall, injections by either method were well tolerated. There were no serious adverse events. Frequency of any local reactogenicity was 16/16 (100%) for Biojector compared to 11/15 (73%) for needle injections. There was no difference in HIV Env-specific antibody response between Biojector and needle delivery. Env-specific antibody responses were more than 10-fold higher in subjects receiving a booster dose of rAd5 vaccine than after a single dose delivered by either method regardless of interval between prime and boost. Conclusions: Biojector delivery did not improve antibody responses to the rAd5 vaccine compared to needle administration. Homologous boosting with rAd5 gene-based vectors can boost insert-specific antibody responses despite pre-existing vector-specific immunity. Trial Registration: Clinicaltrials.gov NCT00709605 [ABSTRACT FROM AUTHOR]

CD8+ T cell exhaustion represents a major hallmark of chronic HIV infection. Two key transcription factors governing CD8+ T cell differentiation, T-bet and Eomesodermin (Eomes), have previously been shown in mice to differentially regulate T cell exhaustion in part through direct modulation of PD-1. Here, we examined the relationship between these transcription factors and the expression of several inhibitory receptors (PD-1, CD160, and 2B4), functional characteristics and memory differentiation of CD8+ T cells in chronic and treated HIV infection. The expression of PD-1, CD160, and 2B4 on total CD8+ T cells was elevated in chronically infected individuals and highly associated with a T-betdimEomeshi expressional profile. Interestingly, both resting and activated HIV-specific CD8+ T cells in chronic infection were almost exclusively T-betdimEomeshi cells, while CMV-specific CD8+ T cells displayed a balanced expression pattern of T-bet and Eomes. The T-betdimEomeshi virus-specific CD8+ T cells did not show features of terminal differentiation, but rather a transitional memory phenotype with poor polyfunctional (effector) characteristics. The transitional and exhausted phenotype of HIV-specific CD8+ T cells was longitudinally related to persistent Eomes expression after antiretroviral therapy (ART) initiation. Strikingly, these characteristics remained stable up to 10 years after ART initiation. This study supports the concept that poor human viral-specific CD8+ T cell functionality is due to an inverse expression balance between T-bet and Eomes, which is not reversed despite long-term viral control through ART. These results aid to explain the inability of HIV-specific CD8+ T cells to control the viral replication post-ART cessation. [ABSTRACT FROM AUTHOR]

The interaction between follicular T helper cells (TFH) and B cells in the lymph nodes and spleen has a major impact on the development of antigen-specific B cell responses during infection or vaccination. Recent studies described a functional equivalent of these cells among circulating CD4 T cells, referred to as peripheral TFH cells. Here, we characterize the phenotype and in vitro B cell helper activity of peripheral TFH populations, as well as the effect of HIV infection on these populations. In co-culture experiments we confirmed CXCR5+ cells from HIV-uninfected donors provide help to B cells and more specifically, we identified a CCR7highCXCR5highCCR6highPD-1high CD4 T cell population that secretes IL-21 and enhances isotype-switched immunoglobulin production. This population is significantly decreased in treatment-naïve, HIV-infected individuals and can be recovered after anti-retroviral therapy. We found impaired immunoglobulin production in co-cultures from HIV-infected individuals and found no correlation between the frequency of peripheral TFH cells and memory B cells, or with neutralization activity in untreated HIV infection in our cohort. Furthermore, we found that within the peripheral TFH population, the expression level of TFH-associated genes more closely resembles a memory, non-TFH population, as opposed to a TFH population. Overall, our data identify a heterogeneous population of circulating CD4 T cells that provides in vitro help to B cells, and challenges the origin of these cells as memory TFH cells. [ABSTRACT FROM AUTHOR]

Background: DNA vaccine immunogenicity has been limited by inefficient delivery. Needle-free delivery of DNA using a CO2-powered Biojector® device was compared to delivery by needle and syringe and evaluated for safety and immunogenicity. Methods: Forty adults, 18–50 years, were randomly assigned to intramuscular (IM) vaccinations with DNA vaccine, VRC-HIVDNA016-00-VP, (weeks 0, 4, 8) by Biojector® 2000™ or needle and syringe (N/S) and boosted IM at week 24 with VRC-HIVADV014-00-VP (rAd5) with N/S at 1010 or 1011 particle units (PU). Equal numbers per assigned schedule had low (≤500) or high (>500) reciprocal titers of preexisting Ad5 neutralizing antibody. Results: 120 DNA and 39 rAd5 injections were given; 36 subjects completed follow-up research sample collections. IFN-γ ELISpot response rates were 17/19 (89%) for Biojector® and 13/17 (76%) for N/S delivery at Week 28 (4 weeks post rAd5 boost). The magnitude of ELISpot response was about 3-fold higher in Biojector® compared to N/S groups. Similar effects on response rates and magnitude were observed for CD8+, but not CD4+ T-cell responses by ICS. Env-specific antibody responses were about 10-fold higher in Biojector-primed subjects. Conclusions: DNA vaccination by Biojector® was well-tolerated and compared to needle injection, primed for greater IFN-γ ELISpot, CD8+ T-cell, and antibody responses after rAd5 boosting. Trial Registration: ClinicalTrials.gov NCT00109629 [ABSTRACT FROM AUTHOR]

There is intense interplay between HIV and the immune system, and the literature is replete with studies describing various immunological phenomena associated with HIV infection. Many of these phenomena seem too broad in scope to be attributable either to HIV-infected celIs or to the HIV-specific immune response. Recently, a more fundamental understanding of how HIV affects various T cells and T cell compartments has emerged. This review covers the role of immune activation in HIV immunopathogenesis, how that activation could be mediated directly by HIV replicating within and damaging the gut mucosal barrier, how HIV affects multiple T cell functions and phenotypes, and how chronic HIV replication induces immune modulatory pathways to negatively regulate certain functions in HIV-specific T cells. [ABSTRACT FROM AUTHOR]

The forces that govern clonal selection during the genesis and maintenance of specific T cell responses are complex, but amenable to decryption by interrogation of constituent clonotypes within the antigen-experienced T cell pools. Here, we used point-mutated peptide-major histocompatibility complex class I (pMHCI) antigens, unbiased TCRB gene usage analysis, and polychromatic flow cytometry to probe directly ex vivo the clonal architecture of antigen-specific CD8+ T cell populations under conditions of persistent exposure to structurally stable virus-derived epitopes. During chronic infection with cytomegalovirus and Epstein-Barr virus, CD8+ T cell responses to immunodominant viral antigens were oligoclonal, highly skewed, and exhibited diverse clonotypic configurations; TCRB CDR3 sequence analysis indicated positive selection at the protein level. Dominant clonotypes demonstrated high intrinsic antigen avidity, defined strictly as a physical parameter, and were preferentially driven toward terminal differentiation in phenotypically heterogeneous populations. In contrast, subdominant clonotypes were characterized by lower intrinsic avidities and proportionately greater dependency on the pMHCI-CD8 interaction for antigen uptake and functional sensitivity. These findings provide evidence that interclonal competition for antigen operates in human T cell populations, while preferential CD8 coreceptor compensation mitigates this process to maintain clonotypic diversity. Vaccine strategies that reconstruct these biological processes could generate T cell populations that mediate optimal delivery of antiviral effector function. [ABSTRACT FROM AUTHOR]

The field of vaccinology began in ignorance of how protection was instilled in vaccine recipients. Today, a greater knowledge of immunology allows us to better understand what is being stimulated by various vaccines that leads to their protective effects: that is, their correlates of protection. Here we describe what is known about the correlates of protection for existing vaccines against a range of different viral diseases and discuss the correlates of protection against disease during natural infection with HIV-1. We will also discuss why it is important to design phase 3 clinical trials of HIV vaccines to determine the correlates of protection for each individual vaccine. [ABSTRACT FROM AUTHOR]

Provides information on providing protective immunity against HIV. Importance of cytotoxic T lymphocytes in immunity; Pathophysiology involved in HIV-1 infection; Oligoclonal expansion of CD8+ T lymphocytes.

Primary HIV-1 infection can be classified into six Fiebig stages based on virological and serological laboratory testing, whereas simian-HIV (SHIV) infection in nonhuman primates (NHPs) is defined in time post-infection, making it difficult to extrapolate NHP experiments to the clinics. We identified and extensively characterized the Fiebig-equivalent stages in NHPs challenged intrarectally or intravenously with SHIVAD8-EO. During the first month post-challenge, intrarectally challenged monkeys were up to 1 week delayed in progression through stages. However, regardless of the challenge route, stages I-II predominated before, and stages V-VI predominated after, peak viremia. Decrease in lymph node (LN) CD4+ T cell frequency and rise in CD8+ T cells occurred at stage V. LN virus-specific CD8+ T cell responses, dominated by degranulation and TNF, were first detected at stage V and increased at stage VI. A similar late elevation in follicular CXCR5+ CD8+ T cells occurred, consistent with higher plasma CXCL13 levels at these stages. LN SHIVAD8-EO RNA+ cells were present at stage II, but appeared to decline at stage VI when virions accumulated in follicles. Fiebig-equivalent staging of SHIVAD8-EO infection revealed concordance of immunological events between intrarectal and intravenous infection despite different infection progressions, and can inform comparisons of NHP studies with clinical data. [ABSTRACT FROM AUTHOR]

HIV infection changes the lymph node (LN) tissue architecture, potentially impairing the immunologic response to antigenic challenge. The tissue-resident immune cell dynamics in virologically suppressed HIV+ patients on combination antiretroviral therapy (cART) are not clear. We obtained LN biopsies before and 10 to 14 days after trivalent seasonal influenza immunization from healthy controls (HCs) and HIV+ volunteers on cART to investigate CD4+ T follicular helper (Tfh) and B cell dynamics by flow cytometry and quantitative imaging analysis. Prior to vaccination, compared with those in HCs, HIV+ LNs exhibited an altered follicular architecture, but harbored higher numbers of Tfh cells and increased IgG+ follicular memory B cells. Moreover, Tfh cell numbers were dependent upon preservation of the follicular dendritic cell (FDC) network and were predictive of the magnitude of the vaccine-induced IgG responses. Interestingly, postvaccination LN samples in HIV+ participants had significantly (P = 0.0179) reduced Tfh cell numbers compared with prevaccination samples, without evidence for peripheral Tfh (pTfh) cell reduction. We conclude that influenza vaccination alters the cellularity of draining LNs of HIV+ persons in conjunction with development of antigen-specific humoral responses. The underlying mechanism of Tfh cell decline warrants further investigation, as it could bear implications for the rational design of HIV vaccines. [ABSTRACT FROM AUTHOR]