Co-culture platform for tuning of cancer receptor density allows for evaluation of bispecific immune cell engagers.

Cancer immunotherapy, where a patient's immune system is harnessed to eradicate cancer cells selectively, is a leading strategy for cancer treatment. However, successes with immune checkpoint inhibitors (ICI) are hampered by reported systemic and organ-specific toxicities and by two-thirds of the patients being non-responders or subsequently acquiring resistance to approved ICIs. Hence substantial efforts are invested in discovering novel targeted immunotherapies aimed at reduced side-effects and improved potency. One way is utilizing the dual targeting feature of bispecific antibodies, which have made them increasingly popular for cancer immunotherapy. Easy and predictive screening methods for activation ranking of candidate drugs in tumor contra non-tumor environments are however lacking. Herein, we present a cell-based assay mimicking the tumor microenvironment by co-culturing B cells with engineered human embryonic kidney 293 T cells (HEK293T), presenting a controllable density of platelet-derived growth factor receptor ß (PDGFRß). A target density panel with three different surface protein levels on HEK293T cells was established by genetic constructs carrying regulatory elements limiting RNA translation of PDGFRß. We employed a bispecific antibody-affibody construct called an AffiMab capable of binding PDGFRß on cancer cells and CD40 expressed by B cells as a model. Specific activation of CD40-mediated signaling of immune cells was demonstrated with the two highest receptor-expressing cell lines, Level 2/3 and Level 4, while low-to-none in the low-expressing cell lines. The concept of receptor tuning and the presented co-culture protocol may be of general utility for assessing and developing novel bi-specific antibodies for immuno-oncology applications.

Izokibep: Preclinical development and first-in-human study of a novel IL-17A neutralizing Affibody molecule in patients with plaque psoriasis.

Psoriasis, an immune-mediated inflammatory disease, affects nearly 125 million people globally. The interleukin (IL)-17A homodimer is a key driver of psoriasis and other autoimmune diseases, including psoriatic arthritis, axial spondyloarthritis, hidradenitis suppurativa, and uveitis. Treatment with monoclonal antibodies (mAbs) against IL-17A provides an improvement in the Psoriasis Area and Severity Index compared to conventional systemic agents. In this study, the AffibodyⓇ technology was used to identify and optimize a novel, small, biological molecule comprising three triple helical affinity domains, izokibep (previously ABY-035), for the inhibition of IL-17A signaling. Preclinical studies show that izokibep, a small 18.6 kDa IL-17 ligand trap comprising two IL-17A-specific Affibody domains and one albumin-binding domain, selectively inhibits human IL-17A in vitro and in vivo with superior potency and efficacy relative to anti-IL-17A mAbs. A Phase 1 first-in-human study was conducted to establish the safety, pharmacokinetics, and preliminary efficacy of izokibep, when administered intravenously and subcutaneously as single doses to healthy subjects, and as single intravenous and multiple subcutaneous doses to patients with psoriasis (NCT02690142; EudraCT No: 2015-004531-13). Izokibep was well tolerated with no meaningful safety concerns identified in healthy volunteers and patients with psoriasis. Rapid efficacy was seen in all psoriasis patients after one dose which further improved in patients receiving multiple doses. A therapeutic decrease in joint pain was also observed in a single patient with concurrent psoriatic arthritis. The study suggests that izokibep has the potential to safely treat IL17A-associated diseases such as psoriasis, psoriatic arthritis, axial spondyloarthritis, hidradenitis suppurativa, and uveitis.

Inhibition of IL17A Using an Affibody Molecule Attenuates Inflammation in ApoE-Deficient Mice.

The balance between pro- and anti-inflammatory cytokines released by immune and non-immune cells plays a decisive role in the progression of atherosclerosis. Interleukin (IL)-17A has been shown to accelerate atherosclerosis. In this study, we investigated the effect on pro-inflammatory mediators and atherosclerosis development of an Affibody molecule that targets IL17A. Affibody molecule neutralizing IL17A, or sham were administered in vitro to human aortic smooth muscle cells (HAoSMCs) and murine NIH/3T3 fibroblasts and in vivo to atherosclerosis-prone, hyperlipidaemic ApoE-/- mice. Levels of mediators of inflammation and development of atherosclerosis were compared between treatments. Exposure of human smooth muscle cells and murine NIH/3T3 fibroblasts in vitro to αIL-17A Affibody molecule markedly reduced IL6 and CXCL1 release in supernatants compared with sham exposure. Treatment of ApoE-/- mice with αIL-17A Affibody molecule significantly reduced plasma protein levels of CXCL1, CCL2, CCL3, HGF, PDGFB, MAP2K6, QDPR, and splenocyte mRNA levels of Ccxl1, Il6, and Ccl20 compared with sham exposure. There was no significant difference in atherosclerosis burden between the groups. In conclusion, administration of αIL17A Affibody molecule reduced levels of pro-inflammatory mediators and attenuated inflammation in ApoE-/- mice.

Evaluation of the Therapeutic Potential of a HER3-Binding Affibody Construct TAM-HER3 in Comparison with a Monoclonal Antibody, Seribantumab.

Human epidermal growth factor receptor type 3 (HER3) is recognized to be involved in resistance to HER-targeting therapies. A number of HER3-targeting monoclonal antibodies are under clinical investigation as potential cancer therapeutics. Smaller high-affinity scaffold proteins are attractive non-Fc containing alternatives to antibodies. A previous study indicated that anti-HER3 affibody molecules could delay the growth of xenografted HER3-positive tumors. Here, we designed a second-generation HER3-targeting construct (TAM-HER3), containing two HER3-specific affibody molecules bridged by an albumin-binding domain (ABD) for extension of blood circulation. Receptor blocking activity was demonstrated in vitro. In mice bearing BxPC-3 xenografts, the therapeutic efficacy of TAM-HER3 was compared to the HER3-specific monoclonal antibody seribantumab (MM-121). TAM-HER3 inhibited heregulin-induced phosphorylation in a panel of HER3-expressing cancer cells and was found to be equally as potent as seribantumab in terms of therapeutic efficacy in vivo and with a similar safety profile. Median survival times were 60 days for TAM-HER3, 54 days for seribantumab, and 41 days for the control group. No pathological changes were observed in cytopathological examination. The multimeric HER3-binding affibody molecule in fusion to ABD seems promising for further evaluation as candidate therapeutics for treatment of HER3-overexpressing tumors.

An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo.

In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor α (sIL-6Rα) and IL-6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, ZIL-6_13 with an affinity (KD) for IL-6 of ∼500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, ZIL-6_13 was fused to either the N- or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira®). One AffiMab construct with ZIL-6_13 positioned at the N-terminus of the heavy chain, denoted ZIL-6_13-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the ZIL-6_13-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.

Engineering of a bispecific affibody molecule towards HER2 and HER3 by addition of an albumin-binding domain allows for affinity purification and in vivo half-life extension.

Emerging strategies in cancer biotherapy include the generation and application of bispecific antibodies, targeting two tumor-associated antigens for improved tumor selectivity and potency. Here, an alternative format for bispecific molecules was designed and investigated, in which two Affibody molecules were linked by an albumin-binding domain (ABD). Affibody molecules are small (6 kDa) affinity proteins and this new format allows for engineering of molecules with similar function as full-length bispecific antibodies, but in a dramatically smaller size (around eight-fold smaller). The ABD was intended to function both as a tag for affinity purification as well as for in vivo half-life extension in future preclinical and clinical investigations. Affinity-purified bispecific Affibody molecules, targeting HER2 and HER3, showed simultaneous binding to the three target proteins (HER2, HER3, and albumin) when investigated in biosensor assays. Moreover, simultaneous interactions with the receptors and albumin were demonstrated using flow cytometry on cancer cells. The bispecific Affibody molecules were also able to block ligand-induced phosphorylation of the HER receptors, indicating an anti-proliferative effect. We believe that this compact and flexible format has great potential for developing new potent bispecific affinity proteins in the future, as it combines the benefits of a small size (e.g. improved tissue penetration and reduced cost of goods) with a long circulatory half-life.

Immunotherapy with an HIV-DNA Vaccine in Children and Adults.

Therapeutic HIV immunization is intended to induce new HIV-specific cellular immune responses and to reduce viral load, possibly permitting extended periods without antiretroviral drugs. A multigene, multi-subtype A, B, C HIV-DNA vaccine (HIVIS) has been used in clinical trials in both children and adults with the aim of improving and broadening the infected individuals' immune responses. Despite the different country locations, different regimens and the necessary variations in assays performed, this is, to our knowledge, the first attempt to compare children's and adults' responses to a particular HIV vaccine. Ten vertically HIV-infected children aged 4-16 years were immunized during antiretroviral therapy (ART). Another ten children were blindly recruited as controls. Both groups continued their antiretroviral treatment during and after vaccinations. Twelve chronically HIV-infected adults were vaccinated, followed by repeated structured therapy interruptions (STI) of their antiretroviral treatment. The adult group included four controls, receiving placebo vaccinations. The HIV-DNA vaccine was generally well tolerated, and no serious adverse events were registered in any group. In the HIV-infected children, an increased specific immune response to Gag and RT proteins was detected by antigen-specific lymphoproliferation. Moreover, the frequency of HIV-specific CD8+ T-cell lymphocytes releasing perforin was significantly higher in the vaccinees than the controls. In the HIV-infected adults, increased CD8+ T-cell responses to Gag, RT and viral protease peptides were detected. No augmentation of HIV-specific lymphoproliferative responses were detected in adults after vaccination. In conclusion, the HIV-DNA vaccine can elicit new HIV-specific cellular immune responses, particularly to Gag antigens, in both HIV-infected children and adults. Vaccinated children mounted transient new HIV-specific immune responses, including both CD4+ T-cell lymphoproliferation and late CD8+ T-cell responses. In the adult cohort, primarily CD8+ T-cell responses related to MHC class I alleles were noted. However, no clinical benefits with respect to viral load reduction were ascribable to the vaccinations alone. No severe adverse effects related to the vaccine were found in either cohort, and no virological failures or drug resistances were detected.

Broad and potent cellular and humoral immune responses after a second late HIV-modified vaccinia virus ankara vaccination in HIV-DNA-primed and HIV-modified vaccinia virus Ankara-boosted Swedish vaccinees.

We have previously shown that an HIV vaccine regimen including three HIV-DNA immunizations and a single HIV-modified vaccinia virus Ankara (MVA) boost was safe and highly immunogenic in Swedish volunteers. A median 38 months after the first HIV-MVA vaccination, 24 volunteers received 10(8) plaque-forming units of HIV-MVA. The vaccine was well tolerated. Two weeks after this HIV-MVA vaccination, 18 (82%) of 22 evaluable vaccinees were interferon (IFN)-γ enzyme-linked immunospot (ELISpot) reactive: 18 to Gag and 10 (45%) to Env. A median minimal epitope count of 4 to Gag or Env was found in a subset of 10 vaccinees. Intracellular cytokine staining revealed CD4(+) and/or CD8(+) T cell responses in 23 (95%) of 24 vaccinees, 19 to Gag and 19 to Env. The frequency of HIV-specific CD4(+) and CD8(+) T cell responses was equally high (75%). A high proportion of CD4(+) and CD8(+) T cell responses to Gag was polyfunctional with production of three or more cytokines (40% and 60%, respectively). Of the Env-specific CD4(+) T cells 40% were polyfunctional. Strong lymphoproliferative responses to Aldrithiol-2 (AT-2)-treated subtype A, B, C, and A_E virus were demonstrable in 21 (95%) of 22 vaccinees. All vaccinees developed binding antibodies to Env and Gag. Neutralizing antibodies were detected in a peripheral blood mononuclear cell (PBMC)-based assay against subtype B and CRF01_AE viruses. The neutralizing antibody response rates were influenced by the vaccine dose and/or mode of delivery used at the previous HIV-MVA vaccination. Thus, a second late HIV-MVA boost induced strong and broad cellular immune responses and improved antibody responses. The data support further exploration of this vaccine concept.

Short-term HIV-1 treatment interruption is associated with dysregulated TLR-stimuli responsiveness.

Viremia during human immunodeficiency virus type-1 (HIV-1) infection results in progressive impairment of several components of the immune system. Here a unique model of repeated treatment interruptions (TIs) was used with the aim to reveal the effect of controlled short-term viremia on innate stimuli responsiveness and circulating dendritic cells (DCs). Sequential peripheral blood samples from HIV-1-infected patients on combination antiretroviral therapy, subjected to repeated TI cycles as part of a therapeutic DNA vaccination study, were analyzed. In vitro responsiveness of peripheral blood mononuclear cells to toll-like receptor (TLR) stimuli was analyzed by cytokine secretion, and frequencies of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were monitored by flow cytometry. These parameters were found not to be significantly different between the vaccinated and placebo groups. Instead, independent of vaccination altered in vitro TLR responsiveness was observed in parallel with TI cycles. TLR7/8-triggered secretion of IL-12 and IFN-α, as well as TLR9-triggered secretion of IL-12, was hyperactivated. In contrast, expression of IFN-α after TLR9 stimulation decreased during the initial cycle of TI. Reduced frequencies of pDCs and mDCs, compared with baseline, were noted before and during the second TI, respectively. Furthermore, spontaneous ex vivo release of IL-12 from PBMC was noted during cycles of TI. In conclusion, these results suggest that consequences of short-term TI include dysregulated TLR responses and fluctuations in the frequencies of circulating DCs. Knowledge of these immunological factors may influence the continuation of stringent treatment schedules during HIV infections.

Inhibiting HER3-mediated tumor cell growth with affibody molecules engineered to low picomolar affinity by position-directed error-prone PCR-like diversification.

The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 pM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

Circulating monocytes are not the major source of plasma cytokines in patients with sepsis.

In sepsis, large quantities of inflammatory cytokines are released into the bloodstream. The cellular source of these cytokines is unclear, and we have here investigated to what extent circulating cells in blood contributed to this production. We used the enzyme-linked immunospot technique to study the spontaneous as well as the lipopolysaccharide (LPS)-induced secretion of the proinflammatory cytokines interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), granulocyte-macrophage colony-stimulating factor, IL-1ß, IL-12p40, and the anti-inflammatory cytokine IL-10 from whole-blood cells. The study comprised 32 septic patients (24 with septic shock) and 30 healthy controls. Despite significantly increased plasma cytokine levels in the septic patients, the number of spontaneous cytokine-secreting cells was small or nonexistent and did not differ between the two groups. Lipopolysaccharide stimulation of cells from the same samples triggered substantially increased numbers of cytokine-producing cells in both patients and controls. However, although the numbers of IL-6- and tumor necrosis factor α-secreting monocytes were very similar in both groups, significantly fewer IL-1ß-, IL-10-, IL-12p40-, and granulocyte-macrophage colony-stimulating factor-secreting monocytes were seen in samples from septic patients as compared with healthy controls. The reduced number of cytokine-secreting cells in response to LPS stimulation correlated with disease severity, as expressed by Sequential Organ Failure Assessment score and the stage of sepsis. In summary, circulating leukocytes did not appear to be responsible for the increased plasma levels of cytokines observed in sepsis. A selective sepsis-induced downregulation of cytokine secretion in response to LPS underscores the complexity of cytokine regulation in sepsis.

The PEDVAC trial: preliminary data from the first therapeutic DNA vaccination in HIV-infected children.

The PEDVAC study is the first trial designed to analyze safety and immunogenicity of a therapeutic vaccination with a multiclade multigene HIV DNA vaccine (HIVIS) in infected children. Twenty HIV-1 vertically infected children (6-16 years of age), on stable antiretroviral treatment for at least 6 months with HIV-1 RNA<50 copies/ml and stable CD4 counts (> 400 cells/mm³ or 25%) over 12 months of follow-up, were recruited into the study. Enrolled patients have been randomized into two arms: a control group of 10 children who continued previous antiretroviral treatment (HAART) (arm A) and a group of 10 children immunized intramuscularly with the HIVIS DNA vaccine in addition to previous HAART (arm B). Immunizations took place at week 0, 4, 12 and the boosting dose is planned at week 36. The 10 children in the vaccine group have received the first 3 priming doses of the HIVIS vaccine. Safety data showed good tolerance to the vaccination schedule. Mild cutaneous self-limeted reactions consisted of local irritation, usually itching or erythema +/- swelling at the injection site, were reported. No severe systemic adverse events have been observed. No vaccinated children had a decrease of CD4 T-cell counts from baseline. None experienced virological failure. Analysis of cellular immune responses was scheduled at week 0, 4, 12, 16, 20, 40, 60, 72 and 96 by standard lymphoproliferation assay, intracellular cytokine staining and cell-ELISA, a miniaturized assay to measure antigen-induced IFNγ secretion. Evaluation of these results is in progress and will provide key information on the status and changes of antigen specific immunity during HIV DNA immunization.

Biodistribution, persistence and lack of integration of a multigene HIV vaccine delivered by needle-free intradermal injection and electroporation.

It is likely that gene-based vaccines will enter the human vaccine area soon. A few veterinary vaccines employing this concept have already been licensed, and a multitude of clinical trials against infectious diseases or different forms of cancer are ongoing. Highly important when developing novel vaccines are the safety aspects and also new adjuvants and delivery techniques needs to be carefully investigated so that they meet all short- and long-term safety requirements. One novel in vivo delivery method for plasmid vaccines is electroporation, which is the application of short pulses of electric current immediately after, and at the site of, an injection of a genetic vaccine. This method has been shown to significantly augment the transfection efficacy and the subsequent vaccine-specific immune responses. However, the dramatic increase in delivery efficacy offered by electroporation has raised concerns of potential increase in the risk of integration of plasmid DNA into the host genome. Here, we demonstrate the safety and lack of integration after immunization with a high dose of a multigene HIV-1 vaccine delivered intradermally using the needle free device Biojector 2000 together with electroporation using Derma Vax™ DNA Vaccine Skin Delivery System. We demonstrate that plasmids persist in the skin at the site of injection for at least four months after immunization. However, no association between plasmid DNA and genomic DNA could be detected as analyzed by qPCR following field inversion gel electrophoresis separating heavy and light DNA fractions. We will shortly initiate a phase I clinical trial in which healthy volunteers will be immunized with this multiplasmid HIV-1 vaccine using a combination of the delivery methods jet-injection and intradermal electroporation.

Skin electroporation: effects on transgene expression, DNA persistence and local tissue environment.

BACKGROUND: Electrical pulses have been used to enhance uptake of molecules into living cells for decades. This technique, often referred to as electroporation, has become an increasingly popular method to enhance in vivo DNA delivery for both gene therapy applications as well as for delivery of vaccines against both infectious diseases and cancer. In vivo electrovaccination (gene delivery followed by electroporation) is currently being investigated in several clinical trials, including DNA delivery to healthy volunteers. However, the mode of action at molecular level is not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: This study investigates intradermal DNA electrovaccination in detail and describes the effects on expression of the vaccine antigen, plasmid persistence and the local tissue environment. Gene profiling of the vaccination site showed that the combination of DNA and electroporation induced a significant up-regulation of pro-inflammatory genes. In vivo imaging of luciferase activity after electrovaccination demonstrated a rapid onset (minutes) and a long duration (months) of transgene expression. However, when the more immunogenic prostate specific antigen (PSA) was co-administered, PSA-specific T cells were induced and concurrently the luciferase expression became undetectable. Electroporation did not affect the long-term persistence of the PSA-expressing plasmid. CONCLUSIONS/SIGNIFICANCE: This study provides important insights to how DNA delivery by intradermal electrovaccination affects the local immunological responses of the skin, transgene expression and clearance of the plasmid. As the described vaccination approach is currently being evaluated in clinical trials, the data provided will be of high significance.

Late administration of plasmid DNA by intradermal electroporation efficiently boosts DNA-primed T and B cell responses to carcinoembryonic antigen.

Heterologous boost immunisation is considered the most efficient way to enhance DNA-primed immune responses. We have previously shown that administration of recombinant carcinoembryonic antigen (CEA) efficiently boosts humoral responses in mice primed with CEA DNA. However, clinical grade recombinant proteins are far more intriguing to produce than plasmid DNA. Therefore, the possibility to use plasmid DNA for both priming and boosting would be beneficial. With the prospect of future use in a clinical trial, we investigated if electroporation-mediated delivery of DNA could be used to boost DNA-primed immune responses to CEA. The Biojector was used to prime BALB/c mice intradermally three times with CEA66 DNA, encoding an intracellular modified form of CEA. Twelve weeks after the last prime, the animals received either one injection of recombinant CEA or one intradermal injection of twtCEA DNA, encoding the wild type CEA fused to a tetanus T helper epitope, in combination with electroporation. Boosting with rCEA protein did not enhance T cell responses to CEA but induced CEA-specific IgG in 4 of 8 mice. In contrast, intradermal delivery of twtCEA DNA by electroporation led to a tenfold increase in IFN-gamma-producing CD8+ T cells, compared to the levels obtained after the third priming immunisation. The DNA boost also induced high CEA-specific IgG titers in all immunised animals (8/8). The data suggests that a late DNA boost, in combination with enhanced DNA delivery by electroporation, could be used to enhance the efficiency of DNA vaccination and substitute for a heterologous protein boost vaccination.

Recombinant Modified Vaccinia Ankara (MVA) effectively boosts DNA-primed HIV-specific immune responses in humans despite pre-existing vaccinia immunity.

The presence of vector-specific immune responses may hamper the induction of responses to a foreign antigen encoded by the vector. We evaluated the impact of pre-existing immunity to vaccinia virus on the induction of HIV-specific responses after immunization of healthy volunteers with a HIV-1 DNA prime-MVA boost vaccine. Following three priming immunizations with HIV-1 DNA plasmids, the volunteers were boosted with a single injection of recombinant MVA encoding HIV-1 proteins. Pre-existing immunity to vaccinia virus did not reduce the proportion of individuals who responded to HIV-1, but did lower the magnitude of responses. Our results suggest that vaccinia-based vectors can be used to efficiently induce immune responses to vectored HIV-1 antigens, even in individuals with pre-existing immunity to vaccinia virus.

Autoimmunogenicity of the helix-loop-helix DNA-binding domain.

Nonimmunogenic character of native DNA, and its high immunogenicity when presented in complex with the DNA-binding proteins indicate that the latter might contain molecular triggers of anti-DNA response. To find if this is the case, we have evaluated the autoimmunogenic potential of the main DNA-binding domain of HIV-1 reverse transcriptase that belongs to the canonical helix-loop-helix type. BALB/c mice were immunized with a peptide representing the domain, alone or in complex with the fragmented human DNA in the presence of an adjuvant. Mice were assessed for specific antibodies, autoantibodies against a panel of self-antigens; glomerular immunoglobulin deposition; and for the signs of autoimmune disease, such as proteinuria, and changes in the blood components. Immunization with the adjuvanted peptide-DNA complex induced autoantibodies against double-stranded DNA, histones, heterochromatin, and kidney proteins; glomerular IgG and IgA deposition; proteinuria; thrombocytopenia, and anemia. Altogether, this identifies the helix-loop-helix DNA-binding domain as one of the molecular triggers of autoimmunity to DNA and DNA-associated proteins. The experiments cast new light on the role of the DNA-binding retroviral proteins in the induction of autoimmunity, and on the origins of autoimmune complications in the microbial infections in general. It also implies that choosing the DNA-binding proteins as vaccine candidates should be done with precaution.

Long-term increase of CD4+ central memory cells in HIV-1-infected individuals by therapeutic HIV-1 rgp160 immunization.

OBJECTIVE: To evaluate functional potential and phenotypic markers in HIV-1-infected patients immunized with HIV-1 rgp160. METHODS: We assessed changes in T-cell phenotype and immune function in 12 HIV-1-infected individuals that were part of a therapeutic vaccine study from 1992 to 1995 [Sandstrom E, Wahren B. Therapeutic immunisation with recombinant gp160 in HIV-1 infection: a randomised double-blind placebo-controlled trial. Nordic VAC-04 Study Group. Lancet 1999;353(9166):1735-42]. The patients received 160 microg HIV-1 rgp160 or placebo i.m. at baseline (day 0), and months 1, 2, 3, 4, 6, and thereafter every 3 months. Frozen peripheral blood mononuclear cells (PBMC) were retrieved from time points 0, 9, 12 and 24 months for phenotypic analysis utilizing flow cytometry. RESULTS: Up-regulation of immune activation markers HLA-DR and CD38 was observed at baseline and throughout the monitoring period on both CD4+ and CD8+ T cells in all patients, reflecting immune activation due to persistent high viral load. Further enhanced expression of activation markers was observed over time in the vaccine group, but not the placebo group. We also observed a consistent long-term increase of the CD4+ central memory population (CD3+CD4+CD45RA-CCR7+) in the vaccinated group. CONCLUSIONS: Administration of eight doses of rgp160 in a year appeared to partially reverse some of the defects exerted by HIV-1 on the immune system. A combination of vaccination with effective antiretroviral therapy (ART) may thus represent an immunotherapeutic intervention for treatment of chronic HIV-1 infection. The improvement of a HIV-1-specific central memory population and HIV-1 antigen-specific CD4+ lymphoproliferative responses may have contributed to the short-term improved survival reported in the vaccinated group.

Challenges of Global Vaccine Development.

The Keystone Symposia conference, Challenges of Global Vaccine Development, attracted approximately 440 scientists, physicians and students from all around the world. The conference goal was to share newly acquired knowledge regarding vaccine-induced immunity, novel technologies, childhood vaccines, preclinical models, as well as correlates of protection. The different sessions and workshops were divided based on these aspects, even though the focus on involving the developing world in the vaccine development pervaded the meeting. Roughly half of the participants represented academia. As a result of generous scholarships, and the fact that the meeting was held in South Africa, at least 23% of the non-US attendants were of African origin. As the number of oral and poster presentations were too numerous to all be covered in this review, selected highlights will be discussed.

Immunization of mice with the nef gene from Human Immunodeficiency Virus type 1: study of immunological memory and long-term toxicology.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Nef, is an attractive vaccine target because it is involved in viral pathogenesis, is expressed early in the viral life cycle and harbors many T and B cell epitopes. Several clinical trials include gene-based vaccines encoding this protein. However, Nef has been shown to transform certain cell types in vitro. Based on these findings we performed a long-term toxicity and immunogenicity study of Nef, encoded either by Modified Vaccinia virus Ankara or by plasmid DNA. BALB/c mice were primed twice with either DNA or MVA encoding Nef and received a homologous or heterologous boost ten months later. In the meantime, the Nef-specific immune responses were monitored and at the time of sacrifice an extensive toxicological evaluation was performed, where presence of tumors and other pathological changes were assessed. RESULTS: The toxicological evaluation showed that immunization with MVAnef is safe and does not cause cellular transformation or other toxicity in somatic organs.Both DNAnef and MVAnef immunized animals developed potent Nef-specific cellular responses that declined to undetectable levels over time, and could readily be boosted after almost one year. This is of particular interest since it shows that plasmid DNA vaccine can also be used as a potent late booster of primed immune responses. We observed qualitative differences between the T cell responses induced by the two different vectors: DNA-encoded nef induced long-lasting CD8+ T cell memory responses, whereas MVA-encoded nef induced CD4+ T cell memory responses. In terms of the humoral immune responses, we show that two injections of MVAnef induce significant anti-Nef titers, while repeated injections of DNAnef do not. A single boost with MVAnef could enhance the antibody response following DNAnef prime to the same level as that observed in animals immunized repeatedly with MVAnef. We also demonstrate the possibility to boost HIV-1 Nef-specific immune responses using the MVAnef construct despite the presence of potent anti-vector immunity. CONCLUSION: This study shows that the nef gene vectored by MVA does not induce malignancies or other adverse effects in mice. Further, we show that when the nef gene is delivered by plasmid or by a viral vector, it elicits potent and long-lasting immune responses and that these responses can be directed towards a CD4+ or a CD8+ T cell response depending on the choice of vector.