A novel peptide-based vaccine candidate with protective efficacy against influenza A in a mouse model.

Current influenza vaccines mainly induce antibody responses to the variable hemagglutinin proteins of the virus strains included in the vaccine. Instead, a broadly protective influenza vaccine should aim at inducing antibody- and/or cell-mediated immunity against conserved viral proteins. Vacc-FLU is a peptide based vaccine combining conserved B and T cell epitopes. Peptide selection was done using a proprietary peptide design platform technology focusing on responses to human leukocyte antigen (HLA)-restricted epitopes. Immunization of wild-type mice and mice transgenic for HLA-A2.1 with the peptide mix successfully induced both humoral and cell mediated immune responses. Partial protection from severe weight loss upon challenge was observed in both mouse strains but was stronger and observed at lower vaccine doses in transgenic mice. Our results show that the Vacc-FLU peptide mix is capable of inducing IFNγ-producing T cells and antibody-producing B cells which can protect from severe disease symptoms upon infection.

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.

Combined effect of Vacc-4x, recombinant human granulocyte macrophage colony-stimulating factor vaccination, and romidepsin on the HIV-1 reservoir (REDUC): a single-arm, phase 1B/2A trial.

BACKGROUND: Immune priming before reversal of latency might be a component of a functional HIV cure. To assess this concept, we assessed if therapeutic HIV immunisation followed by latency reversal would affect measures of viral transcription, plasma viraemia, and reservoir size in patients with HIV on suppressive antiretroviral therapy. METHODS: In this single-arm, phase 1B/2A trial, we recruited adults treated at the Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark (aged ≥18 years) with successfully treated HIV-1 with plasma RNA loads of less than 50 copies per mL for the previous year and CD4 counts of at least 500 cells per µL. Exclusion criteria included CD4 counts of less than 200 cells per µL within the past 2 years, active hepatitis B or C infections, and clinically significant cardiac disease, including QTc prolongation. Participants received six therapeutic intradermal HIV-1 immunisations with 12 mg/mL Vacc-4x and 0·6 mg/mL rhuGM-CSF over 12 weeks (at 0 weeks, 1 week, 2 weeks, 3 weeks, 11 weeks, and 12 weeks) before receiving 5 mg/m(2) intravenous romidepsin once a week for 3 weeks. This procedure was followed by analytical treatment interruption. Coprimary outcomes were changes in copies of HIV-1 DNA (total and integrated) per million CD4 T cells and infectious units per million (IUPM) resting memory CD4 T cells established by viral outgrowth, assessed in all patients receiving at least one dose of active treatment with assessable data. We assessed total HIV-1 DNA at screening, before romidepsin treatment, and 6 weeks after romidepsin treatment. We assessed integrated viral DNA at baseline, before romidepsin treatment, and 8 weeks after romidepsin treatment. We assessed IUPM at screening, 2 weeks before romidepsin treatment, and 6 weeks after romidepsin treatment. This trial is registered at ClinicalTrials.gov, number NCT02092116. FINDINGS: Between May 19, 2014, and Oct 8, 2014, we enrolled 20 individuals, of whom 17 completed all Vacc-4x and rhuGM-CSF administrations and romidepsin infusions. 16 of 17 had assessable total HIV-1 DNA, 15 of 17 had assessable integrated HIV-1 DNA, and six of 17 had assessable IUPM at baseline and at one or more timepoints after study treatment. Total HIV-1 DNA declined from screening to 6 weeks after romidepsin treatment (mean reduction 39·7%, 95% CI -59·7 to -11·5; p=0·012). The decrease in integrated HIV-1 DNA from baseline to 8 weeks after romidepsin treatment was not significant (19·2%, -38·6 to 6·3; p=0·123). Among the six assessable participants, the mean reduction in IUPM from screening to 6 weeks after romidepsin treatment was 38·0% (95% CI -67·0 to -8·0; p=0·019). Of 141 adverse events, 134 (95%) were grade 1 and seven (5%) were grade 2-3. INTERPRETATION: This in-vivo combinatorial approach provides the first evidence for the feasibility of a combined shock and kill strategy, but also emphasises that further optimisation of this strategy is needed to achieve a sizeable effect on the latent reservoir that will translate into clinically measurable benefits for people living with HIV-1. FUNDING: Bionor Pharma, the Research Council of Norway, and SkatteFUNN.

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.

Circular CCR5 peptide conjugates and uses thereof (WO2008074895).

BACKGROUND: Several new strategies targeting HIV infection aim to inhibit virus entry by blocking the chemokine receptor CCR5 used by macrophage tropic strains associated with early infection. The current application uses virus-like particles as a support to present CCR5 peptide antigens. OBJECTIVES: The virus-like particle (VLP)-CCR5 composition aims to function as either a preventative and/or therapeutic vaccine inducing durable autoantibodies that can block CCR5 and prevent HIV entry or attenuate disease progression. METHODS: The novelty of the current application lies in the chemical conjugation of circularised peptide antigens to VLPs, primarily the CCR5 N-terminal domain alone but also including the first extracellular loop (ECL-1). Immunised mice and rabbits generated antibodies that recognised native CCR5 and inhibited entry of pseudotype viruses bearing envelope glycoproteins from diverse primary strains in vitro. RESULTS/CONCLUSIONS: Further work is required to assess the in vivo therapeutic potential of these CCR5 compositions. As therapeutic vaccines and/or preventative vaccines, the potential for selecting CXCR4 tropic virus populations associated with disease progression will need to be considered in addition to the broader consequences of targeting a cellular antigen involved in innate immunity.

Novel peptide-based HIV-1 immunotherapy.

Therapeutic immunisation of human immunodeficiency virus type 1-infected individuals should be actively pursued in the first instance to augment highly active antiretroviral therapy regimens. Peptide-based immunotherapeutic strategies offer considerable advantages over conventional approaches, particularly regarding safety. Peptide design itself is becoming increasingly sophisticated, with the rapid evolution of bioinformatics tools that can analyse not only T cell epitopes, but also their potential for successful presentation on diverse human leukocyte antigen (HLA) class I or II following intracellular processing by antigen-presenting cells (APCs). By targeting conserved viral domains, peptides acquire improved reactivity to diverse viral strains. Dendritic cells represent a powerful route of administration, as they are the most potent APCs and can present exogenous peptides on both HLA class I and II through the process of cross-presentation. In this way, soluble peptides can thereby stimulate both CD4+ and CD8+ T cells.

The endogenous langur type D retrovirus PO-1-Lu and its exogenous counterparts in macaque and langur monkeys.

PO-1-Lu, the endogenous type D retrovirus of langurs (Trachypithecus obscurus) has previously been considered a progenitor to the prototype type D retrovirus, Mason Pfizer monkey virus (M-PMV/SRV-3), that became established in macaque monkeys (Macaca spp.) following a zoonosis. This study reevaluates this hypothesis to include other exogenous SRVs. New sequence information from the gp70(SU)-encoding region of PO-1-Lu shows striking similarity to the newly identified exogenous langur retrovirus, SRV-6, recently isolated from the Hanuman Langur (Semnopithecus entellus). An unrooted, bootstrapped neighbor-joining tree derived from env gene nucleotide sequences shows PO-1-Lu and SRV-6 appear more closely related genetically to SRV-2 than SRV-1 or SRV-3 (M-PMV). This is also reflected in our observations that the M-PMV envelope glycoprotein precursor gPr86(Env) and gp70(SU) were antigenically distinct from PO-1-Lu, although the gp22(TM) glycoproteins were antigenically cross-reactive. The potential that SRV-6 represents an exogenous form of PO-1-Lu that has arisen following a recent zoonosis is discussed.

Membrane transplantation to correct integral membrane protein defects.

In this report we show that the tendency of certain viruses to carry host membrane proteins in their envelopes can be harnessed for transplantation of small patches of plasma membrane, including fully functional, polytopic ion channel proteins and their regulatory binding partners. As a stringent model we tested the topologically complex epithelial ion channel CFTR. Initially an attenuated vaccinia virus was found capable of transferring CFTR in a properly folded, functional and regulatable form to CFTR negative cells. Next we generated viruslike particles (VLPs) composed of retroviral structural proteins that assemble and bud at the host cell plasma membrane. These particles were also shown to mediate functional ion channel transfer. By testing the capacity of complex membrane proteins to incorporate into viral envelopes these experiments provide new insight into the permissiveness of viral envelopment, including the ability of incorporated proteins to retain function and repair defects at the cell surface, and serve as a platform for studies of ion channel and membrane protein biochemistry.

The influence of CD4 and CXCR4 on maedi-visna virus-induced syncytium formation.

CD4 is the principal binding site for human and simian immunodeficiency virus (HIV/SIV) receptor interactions and the a chemokine receptor CXCR4 has been implicated as a primordial lentivirus receptor. This study sought to determine the relevance of CD4 and CXCR4 in virus-receptor interactions for the prototype lentivirus, maedi-visna virus (MVV) of sheep. Neither CD4 nor alpha/beta chemokine receptors represent principal receptors for MVV since human osteosarcoma cells devoid of these molecules were susceptible to productive infection. Interestingly, the presence of either CD4 and/or CXCR4 on indicator cells dramatically enhanced MVV-induced cell fusion (syncytium formation) for three independent virus strains. Syncytium formation results from virus-receptor interactions and can be inhibited by receptor ligands. However, neither SDF-la that binds CXCR4 nor recombinant gp120 (rgp120) that binds CD4 could specifically inhibit the observed enhancement of MVV-induced cell fusion under conditions that significantly reduced HIV-1-induced cell fusion. Our observations suggest that CD4 and CXCR4 may represent optional auxiliary components of an MVV receptor (or receptor complex) that facilitate MVV-mediated membrane fusion events, a feature important for virus entry. This potential accessory role for CXCR4 in MW receptor interactions may reflect the distant relationship between the ovine (MVV) and the human/feline lentiviruses (HIV/FIV).