Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti-HIV-1 drugs.

During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates.

Safety and Immunogenicity of a Randomized Phase 1 Prime-Boost Trial With ALVAC-HIV (vCP205) and Oligomeric Glycoprotein 160 From HIV-1 Strains MN and LAI-2 Adjuvanted in Alum or Polyphosphazene.

BACKGROUND: Prime-boost regimens comprising ALVAC-HIV (prime) and human immunodeficiency virus type 1 (HIV) Env (boost) induce HIV-specific neutralizing antibody and cell-mediated immune responses, but the impact of boost schedule and adjuvant requires further definition. METHODS: A phase 1 trial was conducted. In part A (open label), 19 volunteers received oligomeric glycoprotein 160 from HIV strains MN and LAI-2 (ogp160 MN/LAI-2) with dose escalation (25, 50, 100 µg) and either polyphosphazene (pP) or alum adjuvant. In part B, 72 volunteers received either placebo (n=12) or recombinant canarypox virus expressing HIV antigens (ALVAC-HIV [vCP205]) with different doses and schedules of ogp160 MN/LAI-2 in pP or alum (n = 60). RESULTS: The vaccines were safe and well tolerated, with no vaccine-related serious adverse events. Anti-gp70 V1V2 antibody responses were detected in 17 of 19 part A volunteers (89%) and 10%-100% of part B volunteers. Use of a peripheral blood mononuclear cell-based assay revealed that US-1 primary isolate neutralization was induced in 2 of 19 recipients of ogp160 protein alone (10.5%) and 5 of 49 prime-boost volunteers (10.2%). Among ogp160 recipients, those who received pP were more likely than those who received alum to have serum that neutralized tier 2 viruses (12% vs 0%; P = .015). CONCLUSIONS: Administration of ogp160 with pP induces primary isolate tier 2 neutralizing antibody responses in a small percentage of volunteers, demonstrating proof of concept and underscoring the importance of further optimization of prime-boost strategies for HIV infection prevention. CLINICAL TRIALS REGISTRATION: NCT00004579.

Molecular and Physicochemical Factors Governing Solubility of the HIV gp41 Ectodomain.

The HIV gp41 ectodomain (e-gp41) is an attractive target for the development of vaccines and drugs against HIV because of its crucial role in viral fusion to the host cell. However, because of the high insolubility of e-gp41, most biophysical and structural analyses have relied on the production of truncated versions removing the loop region of gp41 or the utilization of nonphysiological solubilizing conditions. The loop region of gp41 is also known as principal immunodominant domain (PID) because of its high immunogenicity, and it is essential for gp41-mediated HIV fusion. In this study we identify the aggregation-prone regions of the amino acid sequence of the PID and engineer a highly soluble mutant that preserves the trimeric structure of the wild-type e-gp41 under physiological pH. Furthermore, using a reverse mutagenesis approach, we analyze the role of mutated amino acids upon the physicochemical factors that govern solubility of e-gp41. On this basis, we propose a molecular model for e-gp41 self-association, which can guide the production of soluble e-gp41 mutants for future biophysical analyses and biotechnological applications.

Secondary structure analysis of HIV-1-gp41 in solution and adsorbed to aluminum hydroxide by Fourier transform infrared spectroscopy.

The formulation of human vaccines often includes adjuvants such as aluminum hydroxide that are added to enhance the immune responses to vaccine antigens. However, these adjuvants may also affect the conformation of antigenic proteins. Such structural modifications could lead to changes in antigenicity such that suboptimal protective immune responses could be generated relative to those induced by the vaccine antigens alone. Here, we used attenuated total reflectance infrared spectroscopy (ATR-FTIR) to compare the secondary structures of recombinant HIV-1-gp41 (gp41) in solution or adsorbed to aluminum hydroxide. The gp41 secondary structure content was 72% alpha-helices and 28% beta-sheets in 5 mM formate buffer p(2)H 2.5, while it was 66% beta-sheets and 34% random coil in acetonitril/(2)H(2)O (95/5:v/v). A fully reversible conformational change of gp41 in acetonitril/(2)H(2)O (95/5:v/v) was observed upon addition of either 35 mM formate p(2)H 2.5 or 0.1% (w/v) detergent (Tween 20, Hecameg, Brij 35 or beta-d-octyl-glucopyranoside). When gp41 was adsorbed to aluminum hydroxide in the presence of 0.1% (w/v) detergent, in either formate or in acetonitril/(2)H(2)O (95/5:v/v) its secondary structure remained stable and was identical to that of gp41 in 5 mM formate buffer p(2)H 2.5. The method described here could be applied for the characterization of gp41 conformers for use in immunological screening of antigens, and more generally to all antigenic proteins adsorbed to aluminum hydroxide.

Interruption of antiretroviral therapy initiated during primary HIV-1 infection: impact of a therapeutic vaccination strategy combined with interleukin (IL)-2 compared with IL-2 alone in the ANRS 095 Randomized Study.

HIV-specific T cell responses play a critical role in the control of infection. We evaluated the impact of immune-based interventions in patients first treated during primary HIV-1 infection (PHI). Forty-three patients were randomized within three groups, to receive either interleukin-2 (IL-2 group), or boosts of ALVAC-HIV (vCP1433) and LIPO-6T followed by interleukin-2 (Vac-IL2 group), compared with no immune intervention (control group), and were monitored for T cell responses. Impact of strategies on viral replication was subsequently assessed during long-term treatment interruption. HIV-specific CD4(+) T cell responses did not change during the study period in immunized patients relative to controls, and vaccination had only a transient effect on interferon-gamma-producing CD8 responses. Viral rebound after treatment interruption was similar in immunized patients and controls. Forty percent of patients had HIV RNA values <10,000 copies/ml 12 weeks after interruption. The cumulative time off treatment represented almost half the total follow-up period. Immunological and virological status during PHI and HIV DNA load at interruption were predictive of the level of viral rebound after treatment interruption, whereas HIV RNA level during PHI and HIV DNA level at interruption were predictive of the time off treatment. Treatment interruption is safe in patients treated early after primary HIV infection. On the basis of this pilot study, HIV immunizations and interleukin-2 appear to have no supplementary benefit.

Immunological and virological efficacy of a therapeutic immunization combined with interleukin-2 in chronically HIV-1 infected patients.

OBJECTIVE: Several lines of evidence suggest that the immune system may control HIV-1 replication, but that it could fail in the long term. Strategies aimed to elicit specific immune responses may enable patients to contain virus replication. METHODS: HIV-1-infected patients were randomized to continue either their antiviral therapy alone (controls; n = 37) or with four boosts of vaccination combining ALVAC-HIV (vCP1433) and Lipo-6T vaccines (weeks 0, 4, 8, 12) followed by three cycles of subcutaneous interleukin-2 (weeks 16, 24, 32) (Vac-IL-2 group; n = 34). RESULTS: Of the Vac-IL-2 group, 15/32 (47%) exhibited a stable HIV p24 antigen-proliferative response compared with 8/33 (24%) controls (P = 0.049). After vaccination, 19/33 (58%) of the Vac-IL-2 group exhibited a multiepitopic HIV-1-specific CD4 cell proliferative response compared with 9/36 (25%) of controls (P = 0.006). The breadth and the magnitude of HIV-specific interferon-gamma-producing CD8 T cells improved in the Vac-IL-2 group. After stopping antiviral drugs, 24% of the Vac-IL-2 group lowered their viral set point compared with 5% of controls (P = 0.027). Logistic-regression analysis demonstrated that vaccine-elicited immunological responses were predictive of virological control (P = 0.046 and 0.014 for stable and multiepitopic CD4 T cell responses, respectively). CONCLUSION: This study provides proof of the concept that therapeutic immunization before antiviral drug cessation may contribute to the containment of HIV replication.

Assessment of mucosal immunity to HIV-1.

A key gap in the development and evaluation of HIV-1 vaccines is insufficient knowledge with regard to sampling techniques and assessment of mucosal immune responses required for early prevention and inhibition of viral dissemination. In an attempt to start bridging this gap, the EUROPRISE network of scientists working on HIV-1 vaccine and microbicide research organized a workshop with the aim to review the types of mucosal responses/biomarkers currently measured in mucosal immunology and to define how the mucosal responses/biomarkers are measured and/or the assays and sampling methods used. The Workshop addressed two critical questions: first whether, with current knowledge, it would be possible to define a consensus set of mucosal sampling methods to facilitate cross-species comparisons and ensure standardized implementation in clinical trials; second to determine the remaining challenges (technical and logistical) and their possible solutions for assessing mucosal responses to HIV-1 vaccines.

Greater viral rebound and reduced time to resume antiretroviral therapy after therapeutic immunization with the ALVAC-HIV vaccine (vCP1452).

OBJECTIVE: Evaluate immunogenicity and clinical efficacy of two immunization strategies with the ALVAC-HIV-recombinant canarypox vaccine (vCP1452) in treated HIV-infected patients. DESIGN: Randomized, double-blind, placebo-controlled, phase II study of vCP1452 immunization in chronically HIV-infected patients on therapy with CD4 T-cell count more than 350 cells/microl, CD4 nadir less than 400 cells/microl and pHIV-RNA less than 400 copies/ml. Patients were equally randomized to four injections at weeks 0, 4, 8, 20; three injections at weeks 4, 8, 20; and placebo. The primary endpoint was vaccine immunogenicity at week 24 measured by enzyme-linked immunospot-interferon-gamma against the HIV-gag-reverse transcriptase-nef vaccine sequences. Secondary endpoints included time to treatment resumption and viral quantitation following treatment interruption at week 24. Criteria to resume therapy included CD4 T-cell count decline less than 250 cells/microl or 50% decrease from baseline or pHIV-RNA more than 50,000 copies/ml. RESULTS: Sixty-five patients enrolled. Changes from baseline in HIV-specific T cells in the four injection arms (+480 spot-forming cells/M-peripheral blood mononuclear cell) were significant compared to placebo (+8; P = 0.014), but not in the three injection arms (+322). The week 36 pHIV-RNA (log10 copies/ml) after treatment interruption was higher in the four (4.71; P = 0.023) and three (4.82; P = 0.009) injection arms compared to placebo (4.40). Percentages of patients reaching treatment resumption criteria by week 48 were 74, 55 and 23% in the three respective arms (P = 0.013). Two independent factors influenced time to therapy resumption: immunization (hazards ratio = 2.7, P = 0.048 for three injections; hazards ratio = 4.1, P = 0.003 for four injections) and CD4 nadir (hazards ratio = 0.4, P = 0.002). CONCLUSIONS: Significant immunogenicity was induced by vCP1452; however, this strategy was independently associated with a shorter time to resume therapy and higher viral rebound.

Therapeutic immunization with a human immunodeficiency virus (HIV) type 1-recombinant canarypox vaccine in chronically HIV-infected patients: The Vacciter Study (ANRS 094).

This open single-arm study evaluated whether the administration of an HIV-recombinant canarypox vaccine (vCP1433) in highly active antiretroviral therapy (HAART)-treated patients chronically infected with HIV was safe, immunogenic and associated with prolongation of treatment discontinuation: 48 patients received four monthly vCP1433 injections and stopped HAART. Immunization was safe. HIV-p24-specific lymphoproliferative responses (LPR), significantly increased in the whole group after two injections but decreased thereafter, HIV-gag-specific CD8 T cells were boosted in 55% patients tested. Altogether, 11% patients with at least one HIV-specific LPR during immunization remained off therapy after 44 weeks of interruption. Detection of such LPR response at the time of treatment interruption was significantly associated with the probability of remaining off therapy. These results provide rationale for future randomized trials exploring this strategy.

Impact of therapeutic immunization on HIV-1 viremia after discontinuation of antiretroviral therapy initiated during acute infection.

BACKGROUND: Treatment strategies that would induce durable virological control of human immunodeficiency virus (HIV)-1 in the absence of continued antiretroviral therapy (ART) are highly desirable.METHODS. We assessed, in a randomized, double-blind, placebo-controlled trial, whether the addition of therapeutic vaccines (ALVAC-HIV [vCP1452] or ALVAC-HIV and Remune) to ART initiated during acute infection could increase the probability of having a plasma viral load

Long-term memory B-cell responses in recipients of candidate human immunodeficiency virus type 1 vaccines.

The efficacy and practical application of human immunodeficiency virus type 1 (HIV-1) vaccines may depend in part on the longevity of the immune responses generated, particularly those in the memory compartment. Candidate vaccines based on the HIV-1 envelope glycoproteins generate binding and neutralizing antibodies in humans but there have been no prior studies on the long-term persistence and recall of those responses. We evaluated six healthy, HIV non-infected adults who had received a combination of recombinant canarypox HIV-1 vaccines boosted by gp120 and who had achieved a high serum titer of neutralizing antibody to HIV-1 MN. These individuals were administered a gp160 boost 4-5 years after their last vaccination. Four volunteers had detectable binding and neutralizing antibodies at the time of boosting and all six volunteers exhibited a recall binding and neutralizing antibody response. The antibodies neutralized multiple T cell line-adapted (TCLA) strains of virus, including the vaccine strain, but not primary isolates. These results demonstrate that memory B-cell responses can last for many years following HIV-1 envelope glycoprotein immunization. In principle, similar long-term memory may be possible with improved immunogens that generate broadly cross-reactive neutralizing antibodies.

Preparation of clinical-grade recombinant canarypox-human immunodeficiency virus vaccine-loaded human dendritic cells.

Preclinical data are reported that support a human immunodeficiency virus (HIV) vaccine strategy using recombinant canarypox-HIV vectors (ALVAC-HIV) to load human dendritic cells (DCs) with HIV antigens. Clinical-grade DCs were infected with good manufacturing practice-grade ALVAC-HIV vaccine constructs. ALVAC infection, HIV gene expression, and DC viability and function were monitored by use of immunohistochemistry, flow cytometry, blastogenesis assays, antigen-specific interferon (IFN)-gamma enzyme-linked immunospot assay, and enzyme-linked immunosorbent assay protein detection. The vaccines infected both immature and mature DCs, and intracellular HIV-1 Gag protein was detected within hours. ALVAC-HIV induced DC maturation that was mediated by tumor necrosis factor-alpha and induced DC apoptosis that was directly related to the length of vaccine exposure. Of importance, the infected DCs remained functional in T cell stimulation assays and induced HIV antigen-specific CD8(+) T cell production of IFN-gamma from cells of HIV-1-infected individuals. These data support an ongoing HIV vaccine trial comparing conventional vaccine delivery routes with ex vivo vaccine-loaded autologous DCs for immunogenicity in HIV-1-uninfected volunteers.

Safety and immunogenicity of an HIV subtype B and E prime-boost vaccine combination in HIV-negative Thai adults.

ALVAC-HIV (vCP1521) and AIDSVAX B/E were evaluated in a phase 1/2 trial of human immunodeficiency virus (HIV)-negative Thai adults. Of 133 volunteers enrolled, 122 completed the trial. There were no serious vaccine-related adverse events, nor were there intercurrent HIV infections. Lymphoproliferative responses to glycoprotein 120 E were induced in 63% of the volunteers, and HIV-specific CD8 cytotoxic T lymphocyte responses were induced in 24%. Antibody responses increased in frequency and magnitude in association with the dose level of AIDSVAX B/E. Binding and neutralizing antibodies to the MN strain were induced in 100% and 98%, respectively, of the volunteers receiving 600 microg of AIDSVAX B/E, and such antibodies to E strains were induced in 96% and 71%, respectively, of these volunteers. This vaccine combination was well tolerated and was immunogenic, meeting milestones for advancement to phase 3 evaluation.

Comparison between env-specific T-cell epitopic responses in HIV-1-uninfected adults immunized with combination of ALVAC-HIV(vCP205) plus or minus rgp160MN/LAI-2 and HIV-1-infected adults.

In this study, we investigated the CD4 T-helper response induced by ALVAC-HIV(vCP205) +/- rgp160MN/LAI-2 using a series of 15 overlapping amino acid peptides spanning the entire gp160MN/LAI-2 antigen. CD4 Env-specific T-cell lines were established from three groups of HIV-1-negative HIV vaccine recipients: vCP205 + gp160MN/LAI-2, vCP205 only, and gp160MN/LAI-2 only. CD4 Env-specific T-cell lines established from individuals who received the prime-boost vCP205 + rgp160MN/LAI-2 generated strong and broad T-helper responses scattered across the Env sequence, whereas Env-specific T-cell lines from individuals receiving the vCP205 vaccine alone generated reactivity to only a few peptides. CD4 -specific T-cell lines were also established from HIV-1-infected individuals and demonstrated poor reactogenicity to Env peptides in both breadth and amplitude of response. These results highlight the complexity of major histocompatibility complex class II presentation and CD4 antigen-specific reactivity, emphasizing the need to better understand these crucial T-helper cell responses in the setting of HIV infection and HIV vaccine development.

HIV-1 gp41 and gp160 are hyperthermostable proteins in a mesophilic environment. Characterization of gp41 mutants.

HIV gp41(24-157) unfolds cooperatively over the pH range of 1.0-4.0 with T(m) values of > 100 degrees C. At pH 2.8, protein unfolding was 80% reversible and the DeltaH(vH)/DeltaH(cal) ratio of 3.7 is indicative of gp41 being trimeric. No evidence for a monomer-trimer equilibrium in the concentration range of 0.3-36 micro m was obtained by DSC and tryptophan fluorescence. Glycosylation of gp41 was found to have only a marginal impact on the thermal stability. Reduction of the disulfide bond or mutation of both cysteine residues had only a marginal impact on protein stability. There was no cooperative unfolding event in the DSC thermogram of gp160 in NaCl/P(i), pH 7.4, over a temperature range of 8-129 degrees C. When the pH was lowered to 5.5-3.4, a single unfolding event at around 120 degrees C was noted, and three unfolding events at 93.3, 106.4 and 111.8 degrees C were observed at pH 2.8. Differences between gp41 and gp160, and hyperthermostable proteins from thermophile organisms are discussed. A series of gp41 mutants containing single, double, triple or quadruple point mutations were analysed by DSC and CD. The impact of mutations on the protein structure, in the context of generating a gp41 based vaccine antigen that resembles a fusion intermediate state, is discussed. A gp41 mutant, in which three hydrophobic amino acids in the gp41 loop were replaced with charged residues, showed an increased solubility at neutral pH.

Safety and immunogenicity of ALVAC vCP1452 and recombinant gp160 in newly human immunodeficiency virus type 1-infected patients treated with prolonged highly active antiretroviral therapy.

In order to boost immune responses in persons in whom highly active antiretroviral therapy (HAART) was initiated within 120 days of the onset of symptoms of newly acquired human immunodeficiency virus type 1 (HIV-1) infection, we administered vaccines containing a canarypox virus vector, vCP1452, with HIV-1 genes encoding multiple HIV-1 proteins, and recombinant gp160. Fifteen HIV-1-infected subjects who achieved sustained suppression of plasma viremia for at least 2 years were enrolled. While continuing antiretroviral therapy, each subject received at least four intramuscular injections of the vaccines on days 0, 30, 90, and 180. Adverse events were mild, with the most common being transient tenderness at the vCP1452 injection site. Of the 14 patients who completed vaccination, 13 had significant increases in anti-gp120 or anti-p24 antibody titers, and 9 had transient augmentation of their T-cell proliferation responses to gp160 and/or p24. HIV-1-specific CD8(+) T cells were quantified using an intracellular gamma interferon staining assay. Among 11 patients who had increased CD8(+) T-cell responses, seven had responses to more than one HIV-1 antigen. In summary, vaccination with vCP1452 and recombinant gp160 appears safe and immunogenic in newly HIV-1-infected patients on HAART.

Comparison of systemic and mucosal delivery of 2 canarypox virus vaccines expressing either HIV-1 genes or the gene for rabies virus G protein.

BACKGROUND: Since the primary routes of human immunodeficiency type 1 (HIV-1) infection are across mucosal barriers, a randomized trial of canarypox virus-based vectors was conducted in 84 individuals, with delivery of vaccine by mucosal routes, and was accompanied by a detailed analysis of humoral, cellular, and mucosal immune responses. METHODS: Over the course of 6 months, HIV-1-specific (vCP 205) and rabies (vCP 65) canarypox virus vectors were delivered systemically and/or mucosally into the nose, mouth, vagina, or rectum in a 4-dose schedule, followed by 2 doses of HIV-1 MN recombinant glycoprotein (rgp) 120 or subunit rabies vaccine administered by the intramuscular route. RESULTS: Administration of vaccine and collection of samples were well tolerated. Serum IgG HIV-1-specific antibodies to rgp120 were rarely seen after either systemic or mucosal delivery of canarypox virus vaccine. In contrast, serum IgG rabies and canarypox antibodies were detected in all individuals after systemic, but rarely after mucosal, delivery of vaccine. Suggestions of mucosal recognition of HIV-1 antigen included a cytotoxic T lymphocyte response in 4 of 8 individuals after administration of vaccine by the intrarectal route and a limited immunoglobulin A response at the same site. CONCLUSIONS: Each of the routes of vaccine administration was feasible in the context of a phase 1 study with motivated individuals. However, with the doses and routes of administration used, canarypox virus was not an effective mucosal immunogen.