New insights into pathogenesis point to HIV-1 Tat as a key vaccine target.

Despite over 30 years of enormous effort and progress in the field, no preventative and/or therapeutic vaccines against human immunodeficiency virus (HIV) are available. Here, we briefly summarize the vaccine strategies and vaccine candidates that in recent years advanced to efficacy trials with mostly unsatisfactory results. Next, we discuss a novel and somewhat contrarian approach based on biological and epidemiological evidence, which led us to choose the HIV protein Tat for the development of preventive and therapeutic HIV vaccines. Toward this goal, we review here the role of Tat in the virus life cycle as well as experimental and epidemiological evidence supporting its key role in the natural history of HIV infection and comorbidities. We then discuss the preclinical and clinical development of a Tat therapeutic vaccine, which, by improving the functionality and homeostasis of the immune system and by reducing the viral reservoir in virologically suppressed vaccinees, helps to establish key determinants for intensification of combination antiretroviral therapy (cART) and a functional cure. Future developments and potential applications of the Tat therapeutic vaccine are also discussed, as well as the rationale for its use in preventative strategies. We hope this contribution will lead to a reconsideration of the current paradigms for the development of HIV/AIDS vaccines, with a focus on targeting of viral proteins with key roles in HIV pathogenesis.

Mucosal IL-4R antagonist HIV vaccination with SOSIP-gp140 booster can induce high-quality cytotoxic CD4+/CD8+ T cells and humoral responses in macaques.

Inducing humoral, cellular and mucosal immunity is likely to improve the effectiveness of HIV-1 vaccine strategies. Here, we tested a vaccine regimen in pigtail macaques using an intranasal (i.n.) recombinant Fowl Pox Virus (FPV)-gag pol env-IL-4R antagonist prime, intramuscular (i.m.) recombinant Modified Vaccinia Ankara Virus (MVA)-gag pol-IL-4R antagonist boost followed by an i.m SOSIP-gp140 boost. The viral vector-expressed IL-4R antagonist transiently inhibited IL-4/IL-13 signalling at the vaccination site. The SOSIP booster not only induced gp140-specific IgG, ADCC (antibody-dependent cellular cytotoxicity) and some neutralisation activity, but also bolstered the HIV-specific cellular and humoral responses. Specifically, superior sustained systemic and mucosal HIV Gag-specific poly-functional/cytotoxic CD4+ and CD8+ T cells were detected with the IL-4R antagonist adjuvanted strategy compared to the unadjuvanted control. In the systemic compartment elevated Granzyme K expression was linked to CD4+ T cells, whilst Granzyme B/TIA-1 to CD8+ T cells. In contrast, the cytotoxic marker expression by mucosal CD4+ and CD8+ T cells differed according to the mucosal compartment. This vector-based mucosal IL-4R antagonist/SOSIP booster strategy, which promotes cytotoxic mucosal CD4+ T cells at the first line of defence, and cytotoxic CD4+ and CD8+ T cells plus functional antibodies in the blood, may prove valuable in combating mucosal infection with HIV-1 and warrants further investigation.

Design Concepts of Virus-Like Particle-Based HIV-1 Vaccines.

Prophylactic vaccines remain the best approach for controlling the human immunodeficiency virus-1 (HIV-1) transmission. Despite the limited efficacy of the RV144 trial in Thailand, there is still no vaccine candidate that has been proven successful. Consequently, great efforts have been made to improve HIV-1 antigens design and discover delivery platforms for optimal immune elicitation. Owing to immunogenic, structural, and functional diversity, virus-like particles (VLPs) could act as efficient vaccine carriers to display HIV-1 immunogens and provide a variety of HIV-1 vaccine development strategies as well as prime-boost regimes. Here, we describe VLP-based HIV-1 vaccine candidates that have been enrolled in HIV-1 clinical trials and summarize current advances and challenges according to preclinical results obtained from five distinct strategies. This mini-review provides multiple perspectives to help in developing new generations of VLP-based HIV-1 vaccine candidates with better capacity to elicit specific anti-HIV immune responses.

Effective Delivery of Nef-MPER-V3 Fusion Protein Using LDP12 Cell Penetrating Peptide for Development of Preventive/Therapeutic HIV-1 Vaccine.

BACKGROUND: There is no effective and safe preventive/therapeutics vaccine against HIV-1 worldwide. Different viral proteins such as Nef, and two regions of Env including; variable loop of gp120 (V3) and membrane proximal external region of gp41 (MPER) are particularly important for vaccine development in different strategies and they are also the primary targets of cellular and humoral immune responses. On the other side, LDP12 is a new cell-penetrating peptide (CPP) which is capable of therapeutic application and cargoes delivery across the cellular membrane. OBJECTIVE: In current study, we designed and produced Nef-MPER-V3 fusion protein harboring LDP12 that has the capability of being used in future vaccine studies. METHODS: The CPP-protein was expressed in E. coli Rosseta (DE3) strain and purified through Ni-NTA column. Characterization of cellular delivery and toxicity of the recombinant protein were evaluated by western blotting and MTT assay. RESULTS: Our results showed that the CPP-protein was successfully expressed and purified with high yield of 5 mg/L. Furthermore, non-cytotoxic effect was observed and specific band (~ 37 KDa) in western blotting indicated the capability of LDP12 to improve the rate of penetration into HEK-293T cells in comparison with a control sample. CONCLUSION: Altogether, the data indicated that LDP12 CPP could be utilized to internalize HIV-1 Nef-MPER-V3 protein into eukaryotic cell lines without any toxicity and represented a valuable potential vaccine candidate and this guarantees the further evaluation towards the assessment of its immunogenicity in mice, which is currently under process.

Conditional antibody expression to avoid central B cell deletion in humanized HIV-1 vaccine mouse models.

HIV-1 vaccine development aims to elicit broadly neutralizing antibodies (bnAbs) against diverse viral strains. In some HIV-1-infected individuals, bnAbs evolved from precursor antibodies through affinity maturation. To induce bnAbs, a vaccine must mediate a similar antibody maturation process. One way to test a vaccine is to immunize mouse models that express human bnAb precursors and assess whether the vaccine can convert precursor antibodies into bnAbs. A major problem with such mouse models is that bnAb expression often hinders B cell development. Such developmental blocks may be attributed to the unusual properties of bnAb variable regions, such as poly-reactivity and long antigen-binding loops, which are usually under negative selection during primary B cell development. To address this problem, we devised a method to circumvent such B cell developmental blocks by expressing bnAbs conditionally in mature B cells. We validated this method by expressing the unmutated common ancestor (UCA) of the human VRC26 bnAb in transgenic mice. Constitutive expression of the VRC26UCA led to developmental arrest of B cell progenitors in bone marrow; poly-reactivity of the VRC26UCA and poor pairing of the VRC26UCA heavy chain with the mouse surrogate light chain may contribute to this phenotype. The conditional expression strategy bypassed the impediment to VRC26UCA B cell development, enabling the expression of VRC26UCA in mature B cells. This approach should be generally applicable for expressing other bnAbs that are under negative selection during B cell development.

Modified Adenovirus Prime-Protein Boost Clade C HIV Vaccine Strategy Results in Reduced Viral DNA in Blood and Tissues Following Tier 2 SHIV Challenge.

Designing immunogens and improving delivery methods eliciting protective immunity is a paramount goal of HIV vaccine development. A comparative vaccine challenge study was performed in rhesus macaques using clade C HIV Envelope (Env) and SIV Gag antigens. One group was vaccinated using co-immunization with DNA Gag and Env expression plasmids cloned from a single timepoint and trimeric Env gp140 glycoprotein from one of these clones (DNA+Protein). The other group was a prime-boost regimen composed of two replicating simian (SAd7) adenovirus-vectored vaccines expressing Gag and one Env clone from the same timepoint as the DNA+Protein group paired with the same Env gp140 trimer (SAd7+Protein). The env genes were isolated from a single pre-peak neutralization timepoint approximately 1 year post infection in CAP257, an individual with a high degree of neutralization breadth. Both DNA+Protein and SAd7+Protein vaccine strategies elicited significant Env-specific T cell responses, lesser Gag-specific responses, and moderate frequencies of Env-specific TFH cells. Both vaccine modalities readily elicited systemic and mucosal Env-specific IgG but not IgA. There was a higher frequency and magnitude of ADCC activity in the SAd7+Protein than the DNA+Protein arm. All macaques developed moderate Tier 1 heterologous neutralizing antibodies, while neutralization of Tier 1B or Tier 2 viruses was sporadic and found primarily in macaques in the SAd7+Protein group. Neither vaccine approach provided significant protection from viral acquisition against repeated titered mucosal challenges with a heterologous Tier 2 clade C SHIV. However, lymphoid and gut tissues collected at necropsy showed that animals in both vaccine groups each had significantly lower copies of viral DNA in individual tissues compared to levels in controls. In the SAd7+Protein-vaccinated macaques, total and peak PBMC viral DNA were significantly lower compared with controls. Taken together, this heterologous Tier 2 SHIV challenge study shows that combination vaccination with SAd7+Protein was superior to combination DNA+Protein in reducing viral seeding in tissues in the absence of protection from infection, thus emphasizing the priming role of replication-competent SAd7 vector. Despite the absence of correlates of protection, because antibody responses were significantly higher in this vaccine group, we hypothesize that vaccine-elicited antibodies contribute to limiting tissue viral seeding.

Impact of Th1 CD4 Follicular Helper T Cell Skewing on Antibody Responses to an HIV-1 Vaccine in Rhesus Macaques.

Generating durable humoral immunity through vaccination depends upon effective interactions of follicular helper T (Tfh) cells with germinal center (GC) B cells. Th1 polarization of Tfh cells is an important process shaping the success of Tfh-GC B cell interactions by influencing costimulatory and cytokine-dependent Tfh help to B cells. However, the question remains as to whether adjuvant-dependent modulation of Tfh cells enhances HIV-1 vaccine-induced antienvelope (anti-Env) antibody responses. We investigated whether an HIV-1 vaccine platform designed to increase the number of Th1-polarized Tfh cells enhances the magnitude and quality of anti-Env antibodies. Utilizing a novel interferon-induced protein 10 (IP-10)-adjuvanted HIV-1 DNA prime followed by a monophosphoryl lipid A and QS-21 (MPLA+QS-21)-adjuvanted Env protein boost (DIP-10 PALFQ) in macaques, we observed higher anti-Env serum IgG titers with greater cross-clade reactivity, specificity for V1V2, and effector functions than in macaques primed with DNA lacking IP-10 and boosted with MPLA-plus-alum-adjuvanted Env protein (DPALFA) The DIP-10 PALFQ vaccine regimen elicited higher anti-Env IgG1 and lower IgG4 antibody levels in serum, showing for the first time that adjuvants can dramatically impact the IgG subclass profile in macaques. The DIP-10 PALFQ regimen also increased vaginal and rectal IgA antibodies to a greater extent. Within lymph nodes, we observed augmented GC B cell responses and the promotion of Th1 gene expression profiles in GC Tfh cells. The frequency of GC Tfh cells correlated with both the magnitude and avidity of anti-Env serum IgG. Together, these data suggest that adjuvant-induced stimulation of Th1-Tfh cells is an effective strategy for enhancing the magnitude and quality of anti-Env antibody responses.IMPORTANCE The results of the RV144 trial demonstrated that vaccination could prevent HIV transmission in humans and that longevity of anti-Env antibodies may be key to this protection. Efforts to improve upon the prime-boost vaccine regimen used in RV144 have indicated that booster immunizations can increase serum anti-Env antibody titers but only transiently. Poor antibody durability hampers efforts to develop an effective HIV-1 vaccine. This study was designed to identify the specific elements involved in the immunological mechanism necessary to produce robust HIV-1-specific antibodies in rhesus macaques. By clearly defining immune-mediated pathways that improve the magnitude and functionality of the anti-HIV-1 antibody response, we will have the foundation necessary for the rational development of an HIV-1 vaccine.

Presentation of HIV-1 Envelope Trimers on the Surface of Silica Nanoparticles.

Inducing immune responses protecting from HIV infection or at least controlling replication poses a huge challenge to modern vaccinology. An increasingly discussed strategy to elicit a potent and broad neutralizing antibody response is the immobilization of HIV's trimeric envelope (Env) surface receptor on a nanoparticulate carrier. As a conceptual proof, we attached an Env variant (BG505 SOSIP.664) to highly stable and biocompatible silica nanoparticles (SiNPs) via site-specific covalent conjugation or nonspecific adsorption to SiNPs. First, we demonstrated the feasibility of SiNPs as platform for Env presentation by a thorough characterization process during which Env density, attachment stability, and antigenicity were evaluated for both formulations. Binding affinities to selected antibodies were in the low nanomolar range for both formulations confirming that the structural integrity of Env is retained after attachment. Second, we explored the recognition of SiNP conjugates by antigen presenting cells. Here, the uptake of Env attached to SiNPs via a site-specific covalent conjugation was 4.5-fold enhanced, whereas adsorbed Env resulted only in a moderate 1.4-fold increase compared with Env in its soluble form. Thus, we propose SiNPs with site-specifically and covalently conjugated Env preferably in a high density as a promising candidate for further investigations as vaccine platform.

Gene and protein delivery using four cell penetrating peptides for HIV-1 vaccine development.

Cell penetrating peptides (CPPs) can potently transport therapeutic molecules to target cells for treatment of a variety of diseases. Thus, their use is critical to improve therapeutic vaccines. Histidine-rich nona-arginine (HR9) and primary amphipathic peptide (MPG) showed the ability to transfer DNA into the cells. Moreover, the peptide derived from the C-terminal of the tumor suppressor protein p14ARF (M918) and arginine-rich peptide (penetratin) were utilized to deliver polypeptides and proteins into the living cells. In this study, the immunostimulatory properties of HIV-1 Nef DNA and protein constructs were evaluated using small heat shock protein 20 (sHsp20) and Freund's emulsion as an adjuvant, and four CPPs (HR9, MPG, M918, and penetratin) as a gene or protein carrier in BALB/c mice. Our data indicated that the HR9/DNA, MPG/DNA, M918/protein, and penetratin/protein complexes formed the stable nanoparticles that were effectively delivered in HEK-293T cell line at certain ratios. Moreover, a heterologous Hsp20-Nef DNA + MPG prime/rHsp20-Nef protein+M918 boost regimen significantly elicited higher levels of IgG2a, IgG2b, IFN-gamma, and Granzyme B directed toward Th1 responses in a long period (3 months) after the last immunization compared to other groups. Furthermore, the effective role of Hsp20 was detected as a natural adjuvant in enhancing immune responses against HIV-1 Nef antigen. These findings demonstrated that the simultaneous use of M918 and MPG CPPs as protein and gene carriers improves HIV-1 Nef-specific B- and T-cell immune responses as a promising approach for development of HIV-1 monovalent vaccine.

Lentiviral Vector-Based Dendritic Cell Vaccine Suppresses HIV Replication in Humanized Mice.

HIV-1-infected individuals are treated with lifelong antiretroviral drugs to control the infection. A means to strengthen the antiviral T cell response might allow them to control viral loads without antiretroviral drugs. We report the development of a lentiviral vector-based dendritic cell (DC) vaccine in which HIV-1 antigen is co-expressed with CD40 ligand (CD40L) and a soluble, high-affinity programmed cell death 1 (PD-1) dimer. CD40L activates the DCs, whereas PD-1 binds programmed death ligand 1 (PD-L1) to prevent checkpoint activation and strengthen the cytotoxic T lymphocyte (CTL) response. The injection of humanized mice with DCs transduced with vector expressing CD40L and the HIV-1 SL9 epitope induced antigen-specific T cell proliferation and memory differentiation. Upon HIV-1 challenge of vaccinated mice, viral load was suppressed by 2 logs for 6 weeks. Introduction of the soluble PD-1 dimer into a vector that expressed full-length HIV-1 proteins accelerated the antiviral response. The results support development of this approach as a therapeutic vaccine that might allow HIV-1-infected individuals to control virus replication without antiretroviral therapy.

The Extended Impact of Human Immunodeficiency Virus/AIDS Research.

Human immunodeficiency virus (HIV) is one of the most extensively studied viruses in history, and numerous extraordinary scientific advances, including an in-depth understanding of viral biology, pathogenesis, and life-saving antiretroviral therapies, have resulted from investments in HIV/AIDS research. While the substantial investments in HIV/AIDS research are validated solely on these advances, the collateral broader scientific progress resulting from the support of HIV/AIDS research over the past 30 years is extraordinary as well. The positive impact has ranged from innovations in basic immunology and structural biology to treatments for immune-mediated diseases and cancer and has had an enormous effect on the research and public and global health communities well beyond the field of HIV/AIDS. This article highlights a few select examples of the unanticipated and substantial positive spin-offs of HIV/AIDS research on other scientific areas.

TLR3 agonist and CD40-targeting vaccination induces immune responses and reduces HIV-1 reservoirs.

Activation of HIV-1 reservoirs and induction of anti-HIV-1 T cells are critical to control HIV-1 rebound after combined antiretroviral therapy (cART). Here we evaluated in humanized mice (hu-mice) with persistent HIV-1 infection the therapeutic effect of TLR3 agonist and a CD40-targeting HIV-1 vaccine, which consists of a string of 5 highly conserved CD4+ and CD8+ T cell epitope-rich regions of HIV-1 Gag, Nef, and Pol fused to the C-terminus of a recombinant anti-human CD40 antibody (αCD40.HIV5pep). We show that αCD40.HIV5pep vaccination coadministered with poly(I:C) adjuvant induced HIV-1-specific human CD8+ and CD4+ T cell responses in hu-mice. Interestingly, poly(I:C) treatment also reactivated HIV-1 reservoirs. When administrated in therapeutic settings in HIV-1-infected hu-mice under effective cART, αCD40.HIV5pep with poly(I:C) vaccination induced HIV-1-specific CD8+ T cells and reduced the level of cell-associated HIV-1 DNA (or HIV-1 reservoirs) in lymphoid tissues. Most strikingly, the vaccination significantly delayed HIV-1 rebound after cART cessation. In summary, the αCD40.HIV5pep with poly(I:C) vaccination approach both activates replication of HIV-1 reservoirs and enhances the anti-HIV-1 T cell response, leading to a reduced level of cell-associated HIV-1 DNA or reservoirs. Our proof-of-concept study has significant implication for the development of CD40-targeting HIV-1 vaccine to enhance anti-HIV-1 immunity and reduce HIV-1 reservoirs in patients with suppressive cART.

Molecular Signatures of a TLR4 Agonist-Adjuvanted HIV-1 Vaccine Candidate in Humans.

Systems biology approaches have recently provided new insights into the mechanisms of action of human vaccines and adjuvants. Here, we investigated early transcriptional signatures induced in whole blood of healthy subjects following vaccination with a recombinant HIV-1 envelope glycoprotein subunit CN54gp140 adjuvanted with the TLR4 agonist glucopyranosyl lipid adjuvant-aqueous formulation (GLA-AF) and correlated signatures to CN54gp140-specific serum antibody responses. Fourteen healthy volunteers aged 18-45 years were immunized intramuscularly three times at 1-month intervals and whole blood samples were collected at baseline, 6 h, and 1, 3, and 7 days post first immunization. Subtle changes in the transcriptomic profiles were observed following immunization, ranging from over 300 differentially expressed genes (DEGs) at day 1 to nearly 100 DEGs at day 7 following immunization. Functional pathway analysis revealed blood transcription modules (BTMs) related to general cell cycle activation, and innate immune cell activation at early time points, as well as BTMs related to T cells and B cell activation at the later time points post-immunization. Diverse CN54gp140-specific serum antibody responses of the subjects enabled their categorization into high or low responders, at early (<1 month) and late (up to 6 months) time points post vaccination. BTM analyses revealed repression of modules enriched in NK cells, and the mitochondrial electron chain, in individuals with high or sustained antigen-specific antibody responses. However, low responders showed an enhancement of BTMs associated with enrichment in myeloid cells and monocytes as well as integrin cell surface interactions. Flow cytometry analysis of peripheral blood mononuclear cells obtained from the subjects revealed an enhanced frequency of CD56dim NK cells in the majority of vaccines 14 days after vaccination as compared with the baseline. These results emphasize the utility of a systems biology approach to enhance our understanding on the mechanisms of action of TLR4 adjuvanted human vaccines.

Hypothesis tests for stratified mark-specific proportional hazards models with missing covariates, with application to HIV vaccine efficacy trials.

This article develops hypothesis testing procedures for the stratified mark-specific proportional hazards model with missing covariates where the baseline functions may vary with strata. The mark-specific proportional hazards model has been studied to evaluate mark-specific relative risks where the mark is the genetic distance of an infecting HIV sequence to an HIV sequence represented inside the vaccine. This research is motivated by analyzing the RV144 phase 3 HIV vaccine efficacy trial, to understand associations of immune response biomarkers on the mark-specific hazard of HIV infection, where the biomarkers are sampled via a two-phase sampling nested case-control design. We test whether the mark-specific relative risks are unity and how they change with the mark. The developed procedures enable assessment of whether risk of HIV infection with HIV variants close or far from the vaccine sequence are modified by immune responses induced by the HIV vaccine; this question is interesting because vaccine protection occurs through immune responses directed at specific HIV sequences. The test statistics are constructed based on augmented inverse probability weighted complete-case estimators. The asymptotic properties and finite-sample performances of the testing procedures are investigated, demonstrating double-robustness and effectiveness of the predictive auxiliaries to recover efficiency. The finite-sample performance of the proposed tests are examined through a comprehensive simulation study. The methods are applied to the RV144 trial.

Interferon-α Subtypes As an Adjunct Therapeutic Approach for Human Immunodeficiency Virus Functional Cure.

Human immunodeficiency virus (HIV) establishes life-long latency in infected individuals. Although highly active antiretroviral therapy (HAART) has had a significant impact on the course of HIV infection leading to a better long-term outcome, the pool of latent reservoir remains substantial even under HAART. Numerous approaches have been under development with the goal of eradicating the latent HIV reservoir though with limited success. Approaches that combine immune-mediated control of HIV to activate both the innate and the adaptive immune system under suppressive therapy along with "shock and kill" drugs may lead to a better control of the reactivated virus. Interferon-α (IFN-α) is an innate cytokine that has been shown to activate intracellular defenses capable of restricting and controlling HIV. IFN-α, however, harbors numerous functional subtypes that have been reported to display different binding affinities and potency. Recent studies have suggested that certain subtypes such as IFN-α8 and IFN-α14 have potent anti-HIV activity with little or no immune activation, whereas other subtypes such as IFN-α4, IFN-α5, and IFN-α14 activate NK cells. Could these subtypes be used in combination with other strategies to reduce the latent viral reservoir? Here, we review the role of IFN-α subtypes in HIV infection and discuss the possibility that certain subtypes could be potential adjuncts to a "shock and kill" or therapeutic vaccination strategy leading to better control of the latent reservoir and subsequent functional cure.

Postvaccination C-Reactive Protein and C5/gp41732-744 Antibody Level Fold-Changes Over Baseline Are Independent Predictors of Therapeutic HIV Vaccine Effect in a Phase 2 Clinical Study of Vacc-4x.

Therapeutic vaccination has the potential to contribute to functional HIV cure strategies. However, to show functional HIV cure, study participants must be taken off combination antiretroviral therapy (cART). The availability of suitable biomarkers that can predict viral load (VL) or CD4 count outcomes following therapeutic HIV vaccination would reduce the risks associated with cART interruption in such studies. This report sought to determine baseline and postvaccination biomarker predictors of vaccine effect (VE) on VL and CD4 counts following cART interruption in a double-blind, randomized phase 2 study of the peptide-based therapeutic HIV vaccine, Vacc-4x (n = 93), versus placebo (n = 43). Antibody responses to a novel envelope glycoprotein antigen, C5/gp41732-744, and three safety marker measurements [C-reactive protein (CRP), white blood cell, and lactate dehydrogenase] were considered. Interaction tests in univariate and multivariate linear regression models were used to estimate the effect of biomarkers on VE, defined as the VL or CD4 count difference in Vacc-4x versus placebo groups. The reported q-values (considered significant for hypothesis-generating purposes if ≤0.2) accounted for multiple comparisons using the false discovery rate method. Data were analyzed from all available 58 Vacc-4x and 25 placebo recipients before cART resumption. Lower postvaccination fold-change over baseline of CRP concentration (interaction p- (q-) value = 0.005 (0.11) for VL) and higher fold-change of anti-C5/gp41732-744 antibody levels (0.005 (0.11) for VL and 0.009 (0.20) for CD4) were associated with Vacc-4x benefit. These findings suggest potential roles for inflammation and immune activation markers in predicting therapeutic HIV VE.

Improved estimation of the cumulative incidence of rare outcomes.

Studying the incidence of rare events is both scientifically important and statistically challenging. When few events are observed, standard survival analysis estimators behave erratically, particularly if covariate adjustment is necessary. In these settings, it is possible to improve upon existing estimators by considering estimation in a bounded statistical model. This bounded model incorporates existing scientific knowledge about the incidence of an event in the population. Estimators that are guaranteed to agree with existing scientific knowledge on event incidence may exhibit superior behavior relative to estimators that ignore this knowledge. Focusing on the setting of competing risks, we propose estimators of cumulative incidence that are guaranteed to respect a bounded model and show that when few events are observed, the proposed estimators offer improvements over existing estimators in bias and variance. We illustrate the proposed estimators using data from a recent preventive HIV vaccine efficacy trial. Copyright © 2017 John Wiley & Sons, Ltd.

Improving the potency of DNA vaccine encoding HIV-1 Nef antigen using two endogenous adjuvants in mouse model.

BACKGROUND: DNA immunization can induce long-term immune responses, which are required to design an effective HIV vaccine. It was shown that antigen-expressing plasmids can increase the protective immunity against infectious diseases such as: influenza and malaria. However, DNA-based immunizations have poor immunogenicity, thus the use of potent immunoadjuvants can enhance their potency. METHODS: In the current study, preparation of the recombinant HIV-1 Nef, Gp96 and HMGB1 DNA constructs was performed in bacterial system. Then, the immunogenicity of DNA construct harboring HIV-1 Nef gene (pcDNA-Nef) was studied using two endogenous adjuvants (pcDNA-HMGB1 and pcDNA-Gp96) in BALB/c mouse model. RESULTS: Our data showed that co-injection of pcDNA-Nef with pcDNA-HMGB1 effectively raised both humoral and cell-mediated immune responses in mice as compared to pcDNA-Nef adjuvanted with pcDNA-gp96. Indeed, co-immunization of HIV-1 Nef DNA with HMGB1 DNA significantly induced high levels of IgG2a and IFN-γ directed toward Th1 responses and also cytotoxic T lymphocytes (CTLs) activity in comparison with other immunized groups. CONCLUSION: These findings suggest that the full length of HMGB1 gene could be a more efficient adjuvant for improvement of therapeutic HIV DNA-based immunization compared to the full length of gp96 gene (Tab. 1, Fig. 3, Ref. 58).

Cell-Mediated Immune Predictors of Vaccine Effect on Viral Load and CD4 Count in a Phase 2 Therapeutic HIV-1 Vaccine Clinical Trial.

BACKGROUND: In a placebo-controlled trial of the peptide-based therapeutic HIV-1 p24Gag vaccine candidate Vacc-4x, participants on combination antiretroviral therapy (cART) received six immunizations over 18weeks, followed by analytical treatment interruption (ATI) between weeks 28 and 52. Cell-mediated immune responses were investigated as predictors of Vacc-4x effect (VE) on viral load (VL) and CD4 count during ATI. METHODS: All analyses of week 28 responses and fold-changes relative to baseline considered per-protocol participants (Vacc-4x:placebo=72:32) resuming cART after week 40. Linear regression models with interaction tests were used. VE was estimated as the Vacc-4x-placebo difference in log10-transformed VL (VEVL) or CD4 count (VECD4). FINDINGS: A lower fold-change of CD4+ T-cell proliferation was associated with VECD4 at week 48 (p=0.036, multiplicity adjusted q=0.036) and week 52 (p=0.040, q=0.080). A higher fold-change of IFN-γ in proliferation supernatants was associated with VEVL at week 44 (p=0.047, q=0.07). A higher fold-change of TNF-α was associated with VEVL at week 44 (p=0.045, q=0.070), week 48 (p=0.028, q=0.070), and week 52 (p=0.037, q=0.074). A higher fold-change of IL-6 was associated with VEVL at week 48 (p=0.017, q=0.036). TNF-α levels (>median) were associated with VECD4 at week 48 (p=0.009, q=0.009). INTERPRETATION: These exploratory analyses highlight the potential value of investigating biomarkers in T-cell proliferation supernatants for VE in clinical studies.