Antibody 10-1074 suppresses viremia in HIV-1-infected individuals.

Monoclonal antibody 10-1074 targets the V3 glycan supersite on the HIV-1 envelope (Env) protein. It is among the most potent anti-HIV-1 neutralizing antibodies isolated so far. Here we report on its safety and activity in 33 individuals who received a single intravenous infusion of the antibody. 10-1074 was well tolerated and had a half-life of 24.0 d in participants without HIV-1 infection and 12.8 d in individuals with HIV-1 infection. Thirteen individuals with viremia received the highest dose of 30 mg/kg 10-1074. Eleven of these participants were 10-1074-sensitive and showed a rapid decline in viremia by a mean of 1.52 log10 copies/ml. Virologic analysis revealed the emergence of multiple independent 10-1074-resistant viruses in the first weeks after infusion. Emerging escape variants were generally resistant to the related V3-specific antibody PGT121, but remained sensitive to antibodies targeting nonoverlapping epitopes, such as the anti-CD4-binding-site antibodies 3BNC117 and VRC01. The results demonstrate the safety and activity of 10-1074 in humans and support the idea that antibodies targeting the V3 glycan supersite might be useful for the treatment and prevention of HIV-1 infection.

The Rockefeller University Clinical Scholars (KL2) Program 2006-2016.

INTRODUCTION AND METHODS: The Rockefeller Clinical Scholars (KL2) Program began in 1976 and transitioned into a 3-year Master's degree program in 2006 when Rockefeller joined the NIH Clinical and Translational Science Award (CTSA) program. The program consists of ~15 trainees supported by the CTSA KL2 award and University funds. It is designed to provide an optimal environment for junior translational investigators to develop team science and leadership skills by designing and performing a human subjects protocol under the supervision of a distinguished senior investigator mentor and a team of content expert educators. This is complemented by a tutorial focused on important translational skills. RESULTS: Since 2006, 40 Clinical Scholars have graduated from the programs and gone on to careers in academia (72%), government service (5%), industry (15%), and private medical practice (3%); two (5%) remain in training programs. 39/40 remain in translational research careers with 23 NIH awards totaling $23 million, foundation and philanthropic support of $20.3 million, and foreign government and foundation support of $6 million. They have made wide ranging scientific discoveries and have endeavored to translate those discoveries into improved human health. CONCLUSION: The Rockefeller Clinical Scholars (KL2) program provides one model for translational science training.

HIV-1 antibody 3BNC117 suppresses viral rebound in humans during treatment interruption.

Interruption of combination antiretroviral therapy in HIV-1-infected individuals leads to rapid viral rebound. Here we report the results of a phase IIa open label clinical trial evaluating 3BNC117,a broad and potent neutralizing antibody against the CD4 binding site of the HIV-1 Env protein, during analytical treatment interruption in 13 HIV-1-infected individuals. Participants with 3BNC117-sensitive virus outgrowth cultures were enrolled. Results show that two or four 30 mg kg(-1) 3BNC117 infusions,separated by 3 or 2 weeks, respectively, are generally well tolerated.Infusions are associated with a delay in viral rebound of 5-9 weeks after two infusions, and up to 19 weeks after four infusions, or an average of 6.7 and 9.9 weeks, respectively, compared with 2.6 weeks for historical controls (P < 0.00001). Rebound viruses arise predominantly from a single provirus. In most individuals,emerging viruses show increased resistance, indicating escape.However, 30% of participants remained suppressed until antibody concentrations waned below 20 µg ml(-1), and the viruses emerging in all but one of these individuals showed no apparent resistance to 3BCN117, suggesting failure to escape over a period of 9-19 weeks.We conclude that the administration of 3BNC117 exerts strong selective pressure on HIV-1 emerging from latent reservoirs during analytical treatment interruption in humans.

HIV-1 therapy with monoclonal antibody 3BNC117 elicits host immune responses against HIV-1.

3BNC117 is a broad and potent neutralizing antibody to HIV-1 that targets the CD4 binding site on the viral envelope spike. When administered passively, this antibody can prevent infection in animal models and suppress viremia in HIV-1-infected individuals. Here we report that HIV-1 immunotherapy with a single injection of 3BNC117 affects host antibody responses in viremic individuals. In comparison to untreated controls that showed little change in their neutralizing activity over a 6-month period, 3BNC117 infusion significantly improved neutralizing responses to heterologous tier 2 viruses in nearly all study participants. We conclude that 3BNC117-mediated immunotherapy enhances host humoral immunity to HIV-1.

Anti-HA Glycoforms Drive B Cell Affinity Selection and Determine Influenza Vaccine Efficacy.

Protective vaccines elicit high-affinity, neutralizing antibodies by selection of somatically hypermutated B cell antigen receptors (BCR) on immune complexes (ICs). This implicates Fc-Fc receptor (FcR) interactions in affinity maturation, which, in turn, are determined by IgG subclass and Fc glycan composition within ICs. Trivalent influenza virus vaccination elicited regulation of anti-hemagglutinin (HA) IgG subclass and Fc glycans, with abundance of sialylated Fc glycans (sFc) predicting quality of vaccine response. We show that sFcs drive BCR affinity selection by binding the Type-II FcR CD23, thus upregulating the inhibitory FcγRIIB on activated B cells. This elevates the threshold requirement for BCR signaling, resulting in B cell selection for higher affinity BCR. Immunization with sFc HA ICs elicited protective, high-affinity IgGs against the conserved stalk of the HA. These results reveal a novel, endogenous pathway for affinity maturation that can be exploited for eliciting high-affinity, broadly neutralizing antibodies through immunization with sialylated immune complexes.

Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117.

HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg(-1) infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8-2.5 log10 and viraemia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that, as a single agent, 3BNC117 is safe and effective in reducing HIV-1 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.

Dendritic cells induce antigen-specific regulatory T cells that prevent graft versus host disease and persist in mice.

Foxp3(+) regulatory T cells (T reg cells) effectively suppress immunity, but it is not determined if antigen-induced T reg cells (iT reg cells) are able to persist under conditions of inflammation and to stably express the transcription factor Foxp3. We used spleen cells to stimulate the mixed leukocyte reaction (MLR) in the presence of transforming growth factor ß (TGF-ß) and retinoic acid. We found that the CD11c(high) dendritic cell fraction was the most potent at inducing high numbers of alloreactive Foxp3(+) cells. The induced CD4(+)CD25(+)Foxp3(+) cells appeared after extensive proliferation. When purified from the MLR, iT reg cells suppressed both primary and secondary MLR in vitro in an antigen-specific manner. After transfer into allogeneic mice, iT reg cells persisted for 6 mo and prevented graft versus host disease (GVHD) caused by co-transferred CD45RB(hi) T cells. Similar findings were made when iT reg cells were transferred after onset of GVHD. The CNS2 intronic sequence of the Foxp3 gene in the persisting iT reg cells was as demethylated as the corresponding sequence of naturally occurring T reg cells. These results indicate that induced Foxp3(+) T reg cells, after proliferating and differentiating into antigen-specific suppressive T cells, can persist for long periods while suppressing a powerful inflammatory disease.

Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans.

Adjuvants are critical for the success of vaccines. Agonists of microbial pattern recognition receptors (PRRs) are promising new adjuvant candidates. A mechanism through which adjuvants enhance immune responses is to stimulate innate immunity. We studied the innate immune response in humans to synthetic double-stranded RNA (polyinosinic:polycytidylic acid [poly IC] stabilized with poly-L-lysine [poly ICLC]), an agonist for toll-like receptor (TLR) 3, and the cytosolic RNA helicase MDA-5. Transcriptional analysis of blood samples from eight volunteers, after subcutaneous administration of poly ICLC, showed up-regulation of genes involved in multiple innate immune pathways in all subjects, including interferon (IFN) and inflammasome signaling. Blocking type I IFN receptor ex vivo significantly dampened the response to poly IC. Comparative transcriptional analysis showed that several innate immune pathways were similarly induced in volunteers immunized with the highly efficacious yellow fever vaccine. Therefore, a chemically defined PRR agonist like poly ICLC can be a reliable and authentic microbial mimic for inducing innate immune responses in humans.

In vivo electroporation enhances the immunogenicity of an HIV-1 DNA vaccine candidate in healthy volunteers.

BACKGROUND: DNA-based vaccines have been safe but weakly immunogenic in humans to date. METHODS AND FINDINGS: We sought to determine the safety, tolerability, and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly by in vivo electroporation (EP) in a Phase-1, double-blind, randomized placebo-controlled trial in healthy volunteers. Eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline placebo via EP, or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon ELISpot. The number of antigens to which the response was detected improved with EP and increasing dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines. CONCLUSIONS: This is the first demonstration in healthy volunteers that EP is safe, tolerable, and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate. TRIAL REGISTRATION: ClinicalTrials.gov NCT00545987.

Phase 1 safety and immunogenicity evaluation of ADVAX, a multigenic, DNA-based clade C/B' HIV-1 candidate vaccine.

BACKGROUND: We conducted a Phase I dose escalation trial of ADVAX, a DNA-based candidate HIV-1 vaccine expressing Clade C/B' env, gag, pol, nef, and tat genes. Sequences were derived from a prevalent circulating recombinant form in Yunnan, China, an area of high HIV-1 incidence. The objective was to evaluate the safety and immunogenicity of ADVAX in human volunteers. METHODOLOGY/PRINCIPAL FINDINGS: ADVAX or placebo was administered intramuscularly at months 0, 1 and 3 to 45 healthy volunteers not at high risk for HIV-1. Three dosage levels [0.2 mg (low), 1.0 mg (mid), and 4.0 mg (high)] were tested. Twelve volunteers in each dosage group were assigned to receive ADVAX and three to receive placebo in a double-blind design. Subjects were followed for local and systemic reactogenicity, adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA. Cellular immunogenicity was assessed by a validated IFNgamma ELISpot assay and intracellular cytokine staining. ADVAX was safe and well-tolerated, with no vaccine-related serious adverse events. Local and systemic reactogenicity events were reported by 64% and 42% of vaccine recipients, respectively. The majority of events were mild. The IFNgamma ELISpot response rates to any HIV antigen were 0/9 (0%) in the placebo group, 3/12 (25%) in the low-dosage group, 4/12 (33%) in the mid-dosage group, and 2/12 (17%) in the high-dosage group. Overall, responses were generally transient and occurred to each gene product, although volunteers responded to single antigens only. Binding antibodies to gp120 were not detected in any volunteers, and HIV seroconversion did not occur. CONCLUSIONS/SIGNIFICANCE: ADVAX delivered intramuscularly is safe, well-tolerated, and elicits modest but transient cellular immune responses. TRIAL REGISTRATION: Clinicaltrials.gov NCT00249106.

Phase 1 safety and immunogenicity evaluation of ADMVA, a multigenic, modified vaccinia Ankara-HIV-1 B'/C candidate vaccine.

BACKGROUND: We conducted a Phase I dose-escalation trial of ADMVA, a Clade-B'/C-based HIV-1 candidate vaccine expressing env, gag, pol, nef, and tat in a modified vaccinia Ankara viral vector. Sequences were derived from a prevalent circulating HIV-1 recombinant form in Yunnan, China, an area of high HIV incidence. The objective was to evaluate the safety and immunogenicity of ADMVA in human volunteers. METHODOLOGY/PRINCIPAL FINDINGS: ADMVA or placebo was administered intramuscularly at months 0, 1 and 6 to 50 healthy adult volunteers not at high risk for HIV-1. In each dosage group [1x10(7) (low), 5x10(7) (mid), or 2.5x10(8) pfu (high)] volunteers were randomized in a 3:1 ratio to receive ADMVA or placebo in a double-blinded design. Subjects were followed for local and systemic reactogenicity, adverse events including cardiac adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA, immunoflourescent staining, and HIV-1 neutralization. Cellular immunogenicity was assessed by a validated IFNgamma ELISpot assay and intracellular cytokine staining. Anti-vaccinia binding titers were measured by ELISA. ADMVA was generally well-tolerated, with no vaccine-related serious adverse events or cardiac adverse events. Local or systemic reactogenicity events were reported by 77% and 78% of volunteers, respectively. The majority of events were of mild intensity. The IFNgamma ELISpot response rate to any HIV antigen was 0/12 (0%) in the placebo group, 3/12 (25%) in the low dosage group, 6/12 (50%) in the mid dosage group, and 8/13 (62%) in the high dosage group. Responses were often multigenic and occasionally persisted up to one year post vaccination. Antibodies to gp120 were detected in 0/12 (0%), 8/13 (62%), 6/12 (50%) and 10/13 (77%) in the placebo, low, mid, and high dosage groups, respectively. Antibodies persisted up to 12 months after vaccination, with a trend toward agreement with the ability to neutralize HIV-1 SF162 in vitro. Two volunteers mounted antibodies that were able to neutralize clade-matched viruses. CONCLUSIONS/SIGNIFICANCE: ADMVA was well-tolerated and elicited durable humoral and cellular immune responses. TRIAL REGISTRATION: Clinicaltrials.gov NCT00252148.

A randomized therapeutic vaccine trial of canarypox-HIV-pulsed dendritic cells vs. canarypox-HIV alone in HIV-1-infected patients on antiretroviral therapy.

Targeting canarypox (CP)-HIV vaccine to dendritic cells (DCs) elicits anti-HIV-1 immune responses in vitro. We conducted a phase I/II clinical trial to evaluate whether adding DC to a CP-HIV vaccine improved virologic control during analytic treatment interruption (ATI) in HIV-1-infected subjects. Twenty-nine subjects on suppressive antiretroviral therapy were randomized to vaccination with autologous DCs infected with CP-HIV+keyhole limpet hemocyanin (KLH) (arm A, n=14) or CP-HIV+KLH alone (arm B, n=15). The mean viral load (VL) setpoint during ATI did not differ between subjects in arms A and B. A higher percentage of subjects in the DC group had a VL setpoint < 5,000 c/mL during ATI (4/13 or 31% in arm A compared with 0/13 in arm B, p=0.096), but virologic control was transient. Subjects in arm A had a greater increase in KLH lymphoproliferative response than subjects in arm B; however, summed ELISPOT responses to HIV-1 antigens did not differ by treatment arm. We conclude that a DC-CP-HIV vaccine is well-tolerated in HIV-1-infected patients, but does not lower VL setpoint during ATI compared with CP-HIV alone. New methods to enhance the immunogenicity and antiviral efficacy of DC-based vaccines for HIV-1 infection are needed.

The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells.

DNA vaccines promote an immune response by providing antigen-encoding DNA to the recipient, but the efficacy of such vaccines needs improving. Many approaches have considerable potential but currently induce relatively weak immune responses despite multiple high doses of DNA vaccine. Here, we asked whether targeting vaccine antigens to DCs would increase the immunity and protection that result from DNA vaccines. To determine this, we generated a DNA vaccine encoding a fusion protein comprised of the vaccine antigen and a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Following vaccination of mice, the vaccine antigen was expressed selectively by DCs, which were required for the increased efficacy of MHC class I and MHC class II antigen presentation relative to a control scFv DNA vaccine. In addition, a DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein induced 10-fold higher antibody levels and increased numbers of IFN-gamma-producing CD4+ and CD8+ T cells. After a single i.m. injection of the DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein, mice were protected from an airway challenge with a recombinant vaccinia virus expressing the HIV gag p41, even with 1% of the dose of nontargeted DNA vaccine. The efficacy of DNA vaccines therefore may be enhanced by inclusion of sequences such as single-chain antibodies to target the antigen to DCs.

The microbial mimic poly IC induces durable and protective CD4+ T cell immunity together with a dendritic cell targeted vaccine.

CD4(+) Th1 type immunity is implicated in resistance to global infectious diseases. To improve the efficacy of T cell immunity induced by human immunodeficiency virus (HIV) vaccines, we are developing a protein-based approach that directly harnesses the function of dendritic cells (DCs) in intact lymphoid tissues. Vaccine proteins are selectively delivered to DCs by antibodies to DEC-205/CD205, a receptor for antigen presentation. We find that polyriboinosinic:polyribocytidylic acid (poly IC) independently serves as an adjuvant to allow a DC-targeted protein to induce protective CD4(+) T cell responses at a mucosal surface, the airway. After two doses of DEC-targeted, HIV gag p24 along with poly IC, responder CD4(+) T cells have qualitative features that have been correlated with protective function. The T cells simultaneously make IFN-gamma, tumor necrosis factor (TNF)-alpha, and IL-2, and in high amounts for prolonged periods. The T cells also proliferate and continue to secrete IFN-gamma in response to HIV gag p24. The adjuvant role of poly IC requires Toll-like receptor (TLR) 3 and melanoma differentiation-associated gene-5 (MDA5) receptors, but its analog poly IC(12)U requires only TLR3. We suggest that poly IC be tested as an adjuvant with DC-targeted vaccines to induce numerous multifunctional CD4(+) Th1 cells with proliferative capacity.

T cell immune responses to HIV-1.

The recent use of multiparametric flow cytometry to monitor T cell immune responses complements traditional assays, such as IFN-gamma ELISPOT, to provide more information on the functional complexity of CD4+ and CD8+ T cell immune responses induced either by natural infection, or by immunization. In this review, we provide a general background on T cell subsets, and describe the cellular immune response during natural HIV-1 infection. We then review T cell responses to current candidate HIV-1 vaccines. Taken together, this helps to formulate our understanding of the immune correlates of protection required for an effective prophylactic HIV-1 vaccine. Finally, we emphasize current dendritic cell based vaccine strategies designed to modulate immunity to establish immune protection against HIV-1.

Intensified and protective CD4+ T cell immunity in mice with anti-dendritic cell HIV gag fusion antibody vaccine.

Current human immunodeficiency virus (HIV) vaccine approaches emphasize prime boost strategies comprising multiple doses of DNA vaccine and recombinant viral vectors. We are developing a protein-based approach that directly harnesses principles for generating T cell immunity. Vaccine is delivered to maturing dendritic cells in lymphoid tissue by engineering protein antigen into an antibody to DEC-205, a receptor for antigen presentation. Here we characterize the CD4+ T cell immune response to HIV gag and compare efficacy with other vaccine strategies in a single dose. DEC-205-targeted HIV gag p24 or p41 induces stronger CD4+ T cell immunity relative to high doses of gag protein, HIV gag plasmid DNA, or recombinant adenovirus-gag. High frequencies of interferon (IFN)-gamma- and interleukin 2-producing CD4+ T cells are elicited, including double cytokine-producing cells. In addition, the response is broad because the primed mice respond to an array of peptides in different major histocompatibility complex haplotypes. Long-lived T cell memory is observed. After subcutaneous vaccination, CD4+ and IFN-gamma-dependent protection develops to a challenge with recombinant vaccinia-gag virus at a mucosal surface, the airway. We suggest that a DEC-targeted vaccine, in part because of an unusually strong and protective CD4+ T cell response, will improve vaccine efficacy as a stand-alone approach or with other modalities.