Birger Sørensen

Safety and efficacy of the peptide-based therapeutic vaccine for HIV-1, Vacc-4x: a phase 2 randomised, double-blind, placebo-controlled trial.

BACKGROUND Present combination antiretroviral therapy (cART) alone does not cure HIV infection and requires lifelong drug treatment. The potential role of HIV therapeutic vaccines as part of an HIV cure is under consideration. Our aim was to assess the efficacy, safety, and immunogenicity of Vacc-4x, a peptide-based HIV-1 therapeutic vaccine targeting conserved domains on p24(Gag), in adults infected with HIV-1. METHODS Between July, 2008, and June, 2010, we did a multinational double-blind, randomised, phase 2 study comparing Vacc-4x with placebo. Participants were adults infected with HIV-1 who were aged 18-55 years and virologically suppressed on cART (viral load <50 copies per mL) with CD4 cell counts of 400 × 10(6) cells per L or greater. The trial was done at 18 sites in Germany, Italy, Spain, the UK, and the USA. Participants were randomly assigned (2:1) to Vacc-4x or placebo. Group allocation was masked from participants and investigators. Four primary immunisations, weekly for 4 weeks, containing Vacc-4x (or placebo) were given intradermally after administration of adjuvant. Booster immunisations were given at weeks 16 and 18. At week 28, cART was interrupted for up to 24 weeks. The coprimary endpoints were cART resumption and changes in CD4 counts during treatment interruption. Analyses were by modified intention to treat: all participants who received one intervention. Furthermore, safety, viral load, and immunogenicity (as measured by ELISPOT and proliferation assays) were assessed. The 52 week follow-up period was completed in June, 2011. For the coprimary endpoints the proportion of participants who met the criteria for cART resumption was analysed with a logistic regression model with the treatment effect being assessed in a model including country as a covariate. This study is registered with ClinicalTrials.gov, number NCT00659789. FINDINGS 174 individuals were screened; because of slow recruitment, enrolment stopped with 136 of a planned 345 participants and 93 were randomly assigned to receive Vacc-4x and 43 to receive placebo. There were no differences between the two groups for the primary efficacy endpoints in those participants who stopped cART at week 28. Of the participants who resumed cART, 30 (34%) were in the Vacc-4x group and 11 (29%) in the placebo group, and percentage changes in CD4 counts were not significant (mean treatment difference -5·71, 95% CI -13·01 to 1·59). However, a significant difference in viral load was noted for the Vacc-4x group both at week 48 (median 23,100 copies per mL Vacc-4x vs 71,800 copies per mL placebo; p=0·025) and week 52 (median 19,550 copies per mL vs 51,000 copies per mL; p=0·041). One serious adverse event, exacerbation of multiple sclerosis, was reported as possibly related to study treatment. Vacc-4x was immunogenic, inducing proliferative responses in both CD4 and CD8 T-cell populations. INTERPRETATION The proportion of participants resuming cART before end of study and change in CD4 counts during the treatment interruption showed no benefit of vaccination. Vacc-4x was safe, well tolerated, immunogenic, seemed to contribute to a viral-load setpoint reduction after cART interruption, and might be worth consideration in future HIV-cure investigative strategies. FUNDING Norwegian Research Council GLOBVAC Program and Bionor Pharma ASA.

HLA- and dose-dependent immunogenicity of a peptide-based HIV-1 immunotherapy candidate (Vacc-4x).

Objective: The Vacc-4x immunotherapy candidate is composed of four modified peptides corresponding to conserved domains of the HIV-1 protein p24 that preferentially include HLA-A2 restricted elements. Dose-dependent safety and immunogenicity of Vacc-4x and the significance of a HLA-A2 haplotype were examined. Design: Non-AIDS, HIV-1 infected healthy patients (n = 40) stable on HAART with CD4 counts > 300 × 106 cells/l were randomized to receive either low-dose or high-dose Vacc-4x over 26 weeks in an open, prospective phase II clinical trial. Methods: Patients received a total of 10 intradermal injections, using recombinant granulocyte-macrophage colony stimulating factor as a local adjuvant. Vacc-4x-specific cellular responses were monitored in vivo by delayed-type hypersensitivity (DTH) skin test infiltrates and in vitro by both T-cell proliferation, and induction /secretion of cytokines. Results: Most patients developed Vacc-4x-specific DTHs (90%) and proliferative T-cell responses (80%) that were inter-related in magnitude. High-dose Vacc-4x generally induced stronger specific immune responses than low dose in terms of DTH areas and CD4 and CD8 T-cell proliferation. Only HLA-A2 negative patients had a definite dose advantage, and this subgroup had in fact the best overall DTH and proliferative responses. In contrast, no significant dose difference was observed for HLA-A2 positive patients. No serious adverse events were reported. Conclusions: HIV-associated specific responses were safely induced in most patients by Vacc-4x in a dose-dependent manner and were also influenced by the HLA haplotype.

Long-term HIV-specific responses and delayed resumption of antiretroviral therapy after peptide immunization targeting dendritic cells

Long-term HIV-specific immune responses and clinical outcomes were evaluated in HIV-infected patients previously immunized with p24-like peptides (Vacc-4x) targeting dendritic cells (DC). Vacc-4x-induced cellular immune responses were unchanged 1.5 years after completing immunization, and 62% were still off combined antiretroviral treatment (CART). The magnitude of early Vacc-4x responses determined whether the resumption of CART was clinically indicated 2 years after enrolment. These observations encourage further exploration of both Vacc-4x and other HIV peptide-based immunization regimens targeting DC.

Divergent in vitro and in vivo correlates of HIV- specific T-cell responses during onset of HIV viraemia

Background: Cellular immune responses to HIV-1 have been examined mainly in peripheral blood mononuclear cells (PBMC). During onset of HIV replication and antigenaemia after discontinuation of highly active antiretroviral therapy (HAART), PBMC may theoretically contain HIV-specific T cells that are qualitatively and quantitatively different from specific T cells dominating in the tissues. PBMC responses throughout HIV immunotherapy trials may therefore be skewed during recurrent viraemia. Objective: To compare cellular HIV-specific in vitro responses in PBMC during onset of HIV viraemia with corresponding in vivo responses, represented by classical delayed-type hypersensitivity tests (DTH). Methods: HIV patients (n = 38), pre-immunized with four HIV-1 p24-like consensus peptides (Vacc-4x) during HAART, were subjected to a 14-week treatment interruption with recurrent HIV viraemia. Proliferative T-cell responses to Vacc-4x p24 peptides, HIV p24 protein, and cytomegalovirus (CMV) proteins were measured in PBMC. Corresponding Vacc-4x peptide DTH were expressed as skin infiltrate areas after 48 h. Results: After 14 weeks without HAART, HIV-1 RNA increased to 72 500 copies/ml (median). The Vacc-4x p24 peptide- and HIV-1 p24 protein-induced T-cell proliferation concurrently decreased by 81 and 93% in PBMC during viraemia (medians, P ≤ 0.03), whereas proliferative responses to CMV antigens were stable. In contrast, the Vacc-4x DTH areas, rather tended to increase by 36% (P = 0.08) and contained infiltrates dominated by proliferating T cells and macrophages. Conclusions: Divergent in vitro and in vivo HIV-specific cellular immune responses were found during recurrent HIV viraemia. The clinical relevance of both surrogate markers for HIV-related immune responses should be compared in future studies.

Changes in immunoglobulin isotypes and immunoglobulin G (IgG) subclasses during highly active antiretroviral therapy: Anti-p24 IgG1 closely parallels the biphasic decline in plasma viremia

The effects of highly active antiretroviral therapy (HAART) on immunoglobulin isotypes and immunoglobulin G (IgG) subclasses were studied in 12 patients in early stages of HIV-1 infection. Blood samples were obtained at enrollment and 2, 4, 8, 12, 24, 48, and 120 weeks after initiation of HAART. Immunoglobulin concentrations were determined by nephelometry, and anti-p24–specific IgG and IgG1 levels were determined by an enzyme immunoassay. Overall time changes were analyzed in analysis of variance models. IgG and IgG1 levels showed a marked overall decline, whereas other immunoglobulin isotypes and IgG subclasses did not change significantly. Anti-p24–specific IgG1 levels decreased considerably and significantly more in virus isolation–negative patients than in virus isolation–positive patients, as defined according to the ability to isolate HIV-1 from their CD4+ T cells after initiation of therapy. Anti-p24 IgG levels showed a similar but overall weaker decline in the two groups. However, the anti-p24 IgG1 level followed the biphasic decline in plasma viremia more closely than the anti-p24 IgG level, with an initial sharp decline that leveled off with time. These findings suggest that the main reduction in immunoglobulin levels is caused by reduced HIV-1–specific antigen stimulation rather than a general reduction in immune activation. Using anti-p24 IgG1 as a parameter of response to the effect of HAART merits further investigation.

Intranasal Administration of a Therapeutic HIV Vaccine (Vacc-4x) Induces Dose-Dependent Systemic and Mucosal Immune Responses in a Randomized Controlled Trial

Background Vacc-4x, a Gag p24-based therapeutic HIV vaccine, has been shown to reduce viral load set-points after intradermal administration. In this randomized controlled pilot study we investigate intranasal administration of Vacc-4x with Endocine as adjuvant. Methods Safety and immunogenicity were tested in patients on effective ART. They were randomized to low, medium or high dose Vacc-4x or adjuvant alone, administered four times at weekly intervals with no booster. Vacc-4x-specific T cell responses were measured in vitro by proliferation and in vivo by a single DTH skin test at the end of study. Nasal and rectal mucosal secretions were analyzed for Vacc-4x-specific antibodies by ELISA. Immune regulation induced by Vacc-4x was assessed by functional blockade of the regulatory cytokines IL-10 and TGF-β. Results Vacc-4x proliferative T cell responses increased only among the vaccinated (p≤0.031). The low dose group showed the greatest increase in Vacc-4x CD8+T cell responses (p = 0.037) and developed larger DTH (p = 0.005) than the adjuvant group. Rectal (distal) Vacc-4x IgA and IgG antibodies also increased (p = 0.043) in this group. In contrast, the high dose generated higher nasal (local) Vacc-4x IgA (p = 0.028) and serum IgG (p = 0.030) antibodies than the adjuvant. Irrespective of dose, increased Vacc-4x CD4+T cell responses were associated with low proliferation (r = −0.82, p<0.001) and high regulation (r = 0.61, p = 0.010) at baseline. Conclusion Intranasal administration of Vacc-4x with Endocine was safe and induced dose-dependent vaccine-specific T cell responses and both mucosal and systemic humoral responses. The clinical significance of dose, immune regulation and mucosal immunity warrants further investigation. Trial Registration ClinicalTrials.gov NCT01473810

Low frequency of amino acid alterations following therapeutic immunization with HIV-1 Gag p24-like peptides.

Objectives:In chronic HIV-1 infection, the efficacy of a cellular immune response may decline if the virus evolves into variants not recognized by host immune response. The aim of this study was to explore HIV-1 immune escape mutations imposed by therapeutic immunization by investigating sequence variations that might contribute to relapse of viremia in an immunized, HIV-1-infected cohort. Design:We have previously immunized HIV-1-infected individuals on antiretroviral therapy (ART) with a mixture of four short peptides (Vacc-4x) corresponding to p24. Long postimmunization periods without ART allowed longitudinal sequence studies of regions corresponding to Vacc-4x. Methods:Regions of gag p24 including the locations of the Vacc-4x peptides, were sequenced before start of ART, and after postimmunization ART stop (n = 27). Rates and locations of amino acid substitutions were then related to peptide-specific T-cell responses and known epitopes presented by Vacc-4x. Results:The overall rate of amino acid substitutions was low during 35 months (median) of postimmunization viremia, with similar rates of substitution within the regions corresponding to Vacc-4x peptides and other p24 regions despite durable Vacc-4x-specific T-cell responses. Postimmunization amino acid substitutions within Vacc-4x regions were detected in only six patients, and only two of them had measurable T-cell responses against the relevant peptide. Conclusions:The results suggested low prevalence of evolutionary selection of p24 despite new and long-lasting Vacc-4x-specific T-cell responses. The conserved Vacc-4x sequences might therefore be particularly suited for therapeutic immunization. Generally, studies of longitudinal sequence variations after immunization might be valuable when assessing immune escape in HIV vaccine trials.

Restriction fingerprinting and serology in a small outbreak of B15 meningococcal disease among Norwegian soldiers.

In September 1981 a soldier died from meningococcal septicemia in a military camp in Mid-Norway. Soon afterwards one of his room-mates was transferred to a military camp in Northern-Norway where he shared sleeping quarters (room 7D) with 5 other soldiers of whom 2 fell ill with meningococcal disease 1 month later. Throat cultures were obtained from all 128 soldiers at the military camp in Northern-Norway; 41 (32%) harboured meningococci in their throats. The 3 invasive isolates and the isolates from the 4 healthy carriers at room 7D were all group B and type 15 meningococci. However, by DNA fingerprinting we could identify at least 2, probably 3, different individual strains among these 7 isolates. None of these strains were isolated from soldiers outside room 7D. By use of a B15 whole-bacterium ELISA method we showed that the levels of antimeningococcal IgG antibodies in the sera of the two cases at room 7D were low (18 and 28 OD units) compared with the mean IgG levels in the sera of their 4 healthy room mates (1150 OD units) and the mean IgG in the sera from all healthy soldiers (472 OD units).

Boosters of a therapeutic HIV-1 vaccine induce divergent T cell responses related to regulatory mechanisms.

Therapeutic human immunodeficiency virus (HIV) vaccines aim to reduce disease progression by inducing HIV-specific T cells. Vacc-4x are peptides derived from conserved domains within HIV-1 p24 Gag. Previously, Vacc-4x induced T cell responses in 90% of patients which were associated with reduced viral loads. Here we evaluate the effects of Vacc-4x boosters on T cell immunity and immune regulation seven years after primary immunization. Twenty-five patients on effective antiretroviral therapy received two Vacc-4x doses four weeks apart and were followed for 16 weeks. Vacc-4x T cell responses were measured by proliferation (CFSE), INF-γ, CD107a, Granzyme B, Delayed-Type Hypersensitivity test (DTH) and cytokines and chemokines (Luminex). Functional regulation of Vacc-4x-specific T cell proliferation was estimated in vitro using anti-IL-10 and anti-TGF-ß monoclonal antibodies. Vacc-4x-specific CD8(+) T cell proliferation increased in 80% after either the first (64%) or second (16%) booster. Only 40% remained responders after two boosters with permanently increased Vacc-4x-specific proliferative responses (p=0.005) and improved CD8(+) T cell degranulation, IFN-γ production and DTH. At baseline, responders had higher CD8(+) T cell degranulation (p=0.05) and CD4(+) INF-γ production (p=0.01), whereas non-responders had higher production of proinflammatory TNF-α, IL-1α and IL-1ß (p<0.045) and regulatory IL-10 (p=0.07). Notably, IL-10 and TGF-ß mediated downregulation of Vacc-4x-specific CD8(+) T cell proliferation increased only in non-responders (p<0.001). Downregulation during the study correlated to higher PD-1 expression on Vacc-4x-specific CD8(+) T cells (r=0.44, p=0.037), but was inversely correlated to changes in Vacc4x-specific CD8(+) T cell proliferation (r=-0.52, p=0.012). These findings show that Vacc-4x boosters can improve T cell responses in selected patients, but also induce vaccine-specific downregulation of T cell responses in others. Broad surveillance of T cell functions during immunization may help to individualize boosting, where assessment of vaccine-related immune regulation should be further explored as a potential new parameter.

Prospects for HIV-1 therapeutic immunisation and vaccination: the potential contribution of peptide immunogens

Background: Human immunodeficiency virus (HIV)-1 infection continues to challenge the development of antigen-specific immune-based strategies for the management (therapeutic immunisation) and prevention (vaccination) of HIV-1 infection. Objective: This review aims to assess current prospects for HIV-1 therapeutic immunisation with particular emphasis on the contribution of peptide-based immunogens. Methods: The potential for therapeutic immunisation to provide immunological support that can allow for prolonged safe ART-free periods is discussed in light of the Strategies for Management of Antiretroviral Therapy (SMART) study. Different approaches to peptide design are considered including the quality of T-cell responses desired. Results/conclusion: Synthetic peptide immunogens are amenable to modification to improve immunogenicity and reactivity to multiple virus subtypes. Ideally peptide immunogens should incorporate combinations that target restricted, relevant polyfunctional epitopes to regions of HIV-1 associated with control of infection. Peptides showing a beneficial effect following therapeutic immunisation may provide the basis for a future preventative vaccine.