Adalbert Krawczyk

Overcoming drug-resistant herpes simplex virus (HSV) infection by a humanized antibody

Despite the availability of antiviral chemotherapy, herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections remain a severe global health problem. Of particular concern is the growing incidence of drug resistance in immunocompromised patients, which stresses the urgency to develop new effective treatment alternatives. We have developed a humanized monoclonal antibody (mAb hu2c) that completely abrogates viral cell-to-cell spread, a key mechanism by which HSV-1/2 escapes humoral immune surveillance. Moreover, mAb hu2c neutralized HSV fully independent of complement and/or immune effector cell recruitment in a highly efficient manner. Prophylactic and therapeutic administration of mAb hu2c completely prevented infection-related mortality of severely immunodeficient mice being challenged with a lethal dose of HSV-1. The high neutralization capacity of mAb hu2c was fully maintained toward clinical HSV isolates being multiresistant to standard antiviral drugs, and infection was fully resolved in 7/8 nonobese diabetic/SCID mice being infected with a multidrug resistant HSV-1 patient isolate. Immunohistochemical studies revealed no significant cross-reactivity of the antibody toward human tissues. These features warrant further clinical development of mAb hu2c as an immunotherapeutic compound for the management of severe and particularly drug-resistant HSV infections.

Impact of Valency of a Glycoprotein B-Specific Monoclonal Antibody on Neutralization of Herpes Simplex Virus

ABSTRACT Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral neutralization by the monoclonal antibody (MAb) 2c, which specifically recognizes the gB of HSV type 1 (HSV-1) and HSV-2. Binding of MAb 2c to a type-common discontinuous epitope of gB resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by MAb 2c to the recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how MAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent (Fab and scFv) and bivalent [IgG and F(ab′)2] derivatives of MAb 2c. Our data show that the neutralization capacity of MAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of MAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent MAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient NOD/SCID mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific monoclonal antibody prevents HSV-1-induced encephalitis entirely independently from complement activation, antibody-dependent cellular cytotoxicity, and cellular immunity. This indicates the potential for further development of MAb 2c as an anti-HSV drug.

Induction of a robust T- and B-cell immune response in non- and low-responders to conventional vaccination against hepatitis B by using a third generation PreS/S vaccine

Non-responsiveness to conventional hepatitis B vaccines in individuals at high risk of exposure to hepatitis B virus (HBV) is an important public health problem and of particular relevance in health care providers. Yeast-derived conventional HBsAg vaccines fail to induce protective antibody titers in up to 10% of immune competent vaccinees. Therefore, a third generation HBV vaccine, Sci-B-Vac™, was developed which contains in addition to the small S antigen the PreS1 and PreS2 antigens. This vaccine proved to induce a highly potent cellular and humoral immune response in healthy individuals as well as protective antibody levels in non- and low-responders to conventional HBV vaccines. The aim of the study was to examine whether Sci-B-Vac™ triggers cellular and humoral immunity in individuals who failed immunization with conventional vaccines. We immunized 21 volunteers (15 non- and 6 low-responders) according to the standard vaccination schedule (0, 4 and 24 weeks), determined the cellular immunity by proliferation assay and interferon (IFN)-γ ELISpot and measured the anti-HBs antibody titers prior to each vaccination and four weeks after the third vaccine dose. Following three vaccinations, PreS/S-specific T-cell proliferation was detected in 8 out of 15 non-responders and 5 out of 6 low-responders. Specific IFN-γ responses were measured in 2 out of 15 non-responders and 4 out of 6 low-responders. All but one (20/21) study participants developed anti-HBs titers ≥10IU/l after three vaccinations. Anti-HBs ≥100IU/L were detected in 12 out of 15 non-responders and in 6 out of 6 low-responders. Anti-HBs ≥10IU/l and <100IU/l were found in 2 non-responders. These results indicate that Sci-B-Vac™ induces cellular immunity as well as protective anti-HBs antibody titers in non- and low-responders. In conclusion, these results confirm that Sci-B-Vac™ should be administered to non-responders to conventional HBV vaccines and patients with impaired immune function.

A Novel Cardiotropic Murine Adenovirus Representing a Distinct Species of Mastadenoviruses

ABSTRACT During cell culture isolation experiments to recover Dobrava hantavirus from a suspension of liver from a striped field mouse (Apodemus agrarius), an unknown virus was coisolated. Atypically for hantaviruses, it had extensive cytopathic effects. Using a random PCR approach, it was identified as a novel murine adenovirus, MAdV-3 (for MAdV type 3). A plaque-purified virus clone was prepared and further characterized. The complete genome sequence of MAdV-3 was determined to be 30,570 bp in length. Sequence comparisons to other adenovirus species revealed highest similarity to MAdV-1, the representative of the murine adenovirus A species. However, substantial differences were found in the E1, E3, and E4 genomic regions. The phylogenetic distance of MAdV-3 amino acid sequences for pVIII, protease, polymerase, and hexon from MAdV-1 is markedly higher than 0.1 exchange per position, and, based on our cross-neutralization experiments, MAdV-3 and MAdV-1 can be regarded as different serotypes. Therefore, we propose to classify MAdV-3 as the first isolate of a novel adenovirus species, designated murine adenovirus C (MAdV-C). The novel MAdV-3 virus is not only genetically and serologically distinct from MAdV-1 but also shows a unique organ tropism in infected mice. In contrast to MAdV-1, the virus was not detectable in brain but predominantly infected heart tissue. Thus, infection of mice with cardiotropic MAdV-3 might be an interesting animal model of adenovirus-induced myocarditis.

Clinical performance of the novel DiaSorin LIAISON(®) XL murex: HBsAg Quant, HCV-Ab, HIV-Ab/Ag assays.

BACKGROUND The fully automated and closed LIAISON(®)XL platform was developed for reliable detection of infection markers like hepatitis B virus (HBV) surface antigen (HBsAg), hepatitis C virus (HCV) antibodies (Ab) or human immunodeficiency virus (HIV)-Ag/Ab. To date, less is known about the diagnostic performance of this system in direct comparison to the common Abbott ARCHITECT(®) platform. OBJECTIVES We compared the diagnostic performance and usability of the DiaSorin LIAISON(®)XL with the commonly used Abbott ARCHITECT(®) system. STUDY DESIGN The qualitative performance of the above mentioned assays was compared in about 500 sera. Quantitative tests were performed for HBsAg-positive samples from patients under therapy (n=289) and in vitro expressed mutants (n=37). For HCV-Ab, a total number of 155 selected samples from patients chronically infected with different HCV genotypes were tested. RESULTS The concordance between both systems was 99.4% for HBsAg, 98.81% for HCV-Ab, and 99.6% for HIV-Ab/Ag. The quantitative LIAISON(®)XL murex HBsAg assay detected all mutants in comparable amounts to the HBsAg wild type and yielded highly reliable HBsAg kinetics in patients treated with antiviral drugs. Dilution experiments using the 2nd International Standard for HBsAg (WHO) showed a high accuracy of this test. HCV-Ab from patients infected with genotypes 1-3 were equally detected in both systems. Interestingly, S/CO levels of HCV-Ab from patients infected with genotype 3 seem to be relatively low using both systems. CONCLUSIONS The LIAISON(®)XL platform proved to be an excellent system for diagnostics of HBV, HCV, and HIV with equal performance compared to the ARCHITECT(®) system.

Safety and efficacy of REP 2139 and pegylated interferon alfa-2a for treatment-naive patients with chronic hepatitis B virus and hepatitis D virus co-infection (REP 301 and REP 301-LTF): a non-randomised, open-label, phase 2 trial

BACKGROUND REP 2139 clears circulating hepatitis B virus (HBV) surface antigen (HBsAg), enhancing the restoration of functional control of HBV infection by immunotherapy. We assessed the safety and efficacy of REP 2139 and pegylated interferon alfa-2a in patients with chronic HBV and hepatitis D virus (HDV) co-infection. METHODS In this open-label, non-randomised, phase 2 trial, patients aged 18-55 years, who were treatment naive, hepatitis B e antigen [HBeAg] negative, anti-hepatitis D antigen [HDAg] positive, and HDV RNA positive, with serum HBsAg concentrations of more than 1000 IU/mL, and a history of HDV infection for 6 months or more before treatment, were recruited at Toma Ciorbă Hospital of Infectious Diseases in Chișinău, Moldova. Patients were excluded if they had HDV superinfection, liver infections other than HBV and HDV, or liver cirrhosis. Patients received 500 mg intravenous REP 2139 once per week for 15 weeks, followed by combined therapy with 250 mg intravenous REP 2139 and 180 μg subcutaneous pegylated interferon alfa-2a once per week for 15 weeks, then monotherapy with 180 μg pegylated interferon alfa-2a once per week for 33 weeks. The primary endpoints assessed at the end of treatment were the safety and tolerability of the treatment regimen, analysed in the intention-to-treat population. Secondary outcomes included the proportion of patients with serum HBsAg less than 50 IU/mL, the proportion of patients with suppressed HBV DNA, and the proportion of patients who maintained these responses through follow-up. The REP 301 trial is registered with ClinicalTrials.gov, number NCT02233075. We also did an additional follow-up at 1 year after the end of treatment, as an interim analysis of the REP 301-LTF trial (planned duration 3 years), registered with ClinicalTrials.gov, number NCT02876419, which is ongoing but not recruiting patients. FINDINGS Between Sept 8, 2014, and Jan 27, 2015, we enrolled 12 patients into the REP 301 study. All 12 patients experienced at least one adverse event during treatment: two (17%) patients experienced anaemia, eight (67%) neutropenia, and ten (83%) thrombocytopenia. Five (42%) patients had raised alanine aminotransferase levels, four (33%) had raised aspartate aminotransferase levels, and two (17%) had increased bilirubin concentrations. Four (33%) patients had a serious adverse event, and 12 (100%) patients had treatment-emergent lab abnormalities. Six patients had HBsAg levels less than 50 IU/mL by the end of treatment (all <0·05 IU/mL); five maintained this level of suppression at the end of 1 year follow-up. Six patients had hepatitis B surface antibody (anti-HBs) titres above 10 mIU/mL at the end of treatment (five had maximum anti-HBs concentrations of 7681-86 532 mIU/mL during treatment), which were maintained at the end of 1 year follow-up in these five patients. Elevated alanine and aspartate aminotransferase concentrations and profound elevations of anti-HBs titres were restricted to patients who had HBsAg levels of less than <1 IU/mL before the introduction of pegylated interferon alfa-2a. Nine patients had suppressed HBV DNA (<10 IU/mL]) at the end of treatment, which was maintained by seven patients and newly established in an eighth patient at the end of 1 year follow-up. 11 patients became HDV RNA negative during treatment, with nine remaining HDV RNA negative at the end of treatment; seven of these patients remained HDV RNA negative by the end of 1 year follow-up. By the end of 1 year follow-up, normalisation of serum aminotransferases occurred in nine of 12 patients. INTERPRETATION Combined REP 2139 and pegylated interferon alfa-2a therapy is safe, well tolerated, and establishes functional control of HBV and HDV co-infection and normalisation of serum aminotransferases in a high proportion of patients 1 year after therapy. This combination therapy approach might provide a new treatment option for patients with HBV and HDV co-infection. FUNDING Replicor.

Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans.

Therapeutic vaccination of a liver transplant recipient suffering from fulminant hepatitis B during pregnancy.

We report a case of successful therapeutic vaccination of a liver transplant recipient with nonresponder characteristics with a third-generation hepatitis B virus (HBV) vaccine. A 40-year-old pregnant woman presented to our hospital in 2009 with fulminant hepatitis requiring assisted mechanical ventilation and catecholamines. Laboratory tests showed elevated transaminase (glutamic oxaloacetic transaminase level 5 1.507 U/L, glutamic pyruvic transaminase level 5 1.468 U/L) and bilirubin levels (3.68 mg/dL) and decreased liver synthesis (Quick’s value 5 49%). An acute HBV infection was diagnosed with positive findings for hepatitis B surface (HBs) antigen, hepatitis B e (HBe) antigen, and antibody to hepatitis B core (HBc) antigen immunoglobulin M and an HBV DNA level of 6 3 10 copies/mL. Abdominal ultrasound revealed splenomegaly. The initial treatment consisted of lamivudine (100 mg/day), cefazolin, and vitamin K. Because of the rapid deterioration of her liver function (Quick’s value 5 24%), a prothrombin concentrate was applied, and a caesarean section was performed. To prevent mother-to-infant transmission of HBV, polyclonal hepatitis B immunoglobulin (HBIG) was administrated in combination with a standard HBV vaccine to the newborn. Two days after her hospitalization, the patient developed liver and kidney failure and received an orthotopic liver graft from a deceased donor. Immunosuppression was established with tacrolimus, mycophenolate mofetil, and prednisone. To maintain an antibody to hepatitis B surface antigen (anti-HBs) titer higher than 100 IU/L and thus reduce the risk of endogenous HBV reinfection, lamivudine treatment (100 mg/day) was continued in combination with HBIG (500 IU weekly) for 3 years. The patient recovered within 2 weeks and was discharged from the hospital. Immunosuppressive and antiviral therapies were continued for 3 years. The patient remained at high risk for reinfection because of exposure to her chronically HBV-infected partner. Vaccination with a conventional HBV vaccine, which had been successful in prior studies, held only a minor promise of success because (1) the patient displayed human leukocyte antigen DRB1*03 and DQB1*02 (2 antigens associated with an absent humoral immune response to a hepatitis B surface antigen vaccine), and (2) she had a body mass index of 36.8 kg/m. Because the patient was unable to produce sufficient amounts of anti-HBs antibodies, therapeutic vaccination was started in June 2012 with the novel, thirdgeneration HBV vaccine Sci-B-Vac (SciGen, Ltd., Gateway East, Singapore), which had previously shown encouraging results in healthy individuals, nonresponders, and liver transplant recipients. Remaining on HBIG prophylaxis and immunosuppression, the patient was vaccinated at weeks 0, 1, 3, 5 and 7. Although this vaccination allowed a considerable reduction in the frequency of HBIG application, no increase in anti-HBs levels was observed, and cellular immunity was detected only intermittently (Fig. 1). Three months after the last vaccination, the patient began to receive 3 additional doses of Sci-B-Vac at monthly intervals. The antiHBs titer increased up to 1179 IU/L, and this suggested effective protection from HBV infection. In parallel, HBV-specific lymphocyte proliferation reached a stimulation index of 8.7, and there was an HBV-specific enzyme-linked immunosorbent spot assay response as high as 57 interferon-g spots per 4 3 10 cells. Thus, the prophylactic application of HBIG was stopped 3

Assessing the risk of CMV reactivation and reconstitution of antiviral immune response post bone marrow transplantation by the QuantiFERON-CMV-assay and real time PCR

BACKGROUND CMV reactivation is a major cause of severe complications in allogeneic hematopoietic stem cell transplant (HSCT) recipients. The risk of CMV reactivation depends on the serostatus (+/-) of the donor (D) and recipient (R). The reconstitution of CMV-specific T-cell responses after transplantation is crucial for the control of CMV reactivation. OBJECTIVES The study aimed to determine the cellular immune status correlating with protection from high-level CMV viremia (>5000 copies/ml) and disease. STUDY DESIGN We monitored CMV-specific cellular immune responses in 9 high-risk (D-/R+), 14 intermediate risk (D+/R+) and 3 low risk individuals (D+/R-), and 8 CMV negative controls (D-/R-). Interferon- γ (IFN-γ) levels as a marker for the CD8+ T-cell response were determined by the QuantiFERON-CMV-assay and compared to viral loads determined by PCR. RESULTS Early CMV reactivation was detected in all high-risk and 13/14 intermediate risk individuals. High-level viremia was detected in 5/7 high and 7/14 intermediate risk patients. Reconstitution of the CMV-specific cellular immune response started from 3 months after transplantation and resulted in protection against CMV reactivation. Re-establishing of CMV-specific T-cell immune responses with IFN- γ levels >8.9 IU/ml is crucial for protection from high-level CMV viremia. CONCLUSIONS Monitoring of HSCT-recipients with the QuantiFERON-CMV-assay might be of great benefit to optimize antiviral treatment.

A Therapeutic Antiviral Antibody Inhibits the Anterograde Directed Neuron-to-Cell Spread of Herpes Simplex Virus and Protects against Ocular Disease

Herpes simplex virus (HSV) is a leading cause of blindness and viral encephalitis in the developed world. Upon reactivation from sensory neurons, HSV returns via axonal transport to peripheral tissues where it causes, e.g., severe, potentially blinding ocular diseases. In the present study we investigated whether the HSV-1/2 glycoprotein B-specific antibody mAb 2c or its humanized counterpart mAb hu2c can protect from ocular disease in a mouse model of HSV-1-induced acute retinal necrosis (ARN). In this model the viral spread from the initially infected to the contralateral eye resembles the routes taken in humans upon HSV reactivation. Systemic antibody treatment prior or early after infection effectively protected the mice from the development of ARN. These observations suggest that the antibody potently neutralized the infection and inhibited the viral transmission, since there was almost no virus detectable in the contralateral eyes and trigeminal ganglia of antibody treated mice. Besides of neutralizing free virus or limiting the infection via activating the complement or cellular effector functions, blocking of the anterograde directed neuron-to-cell spread of HSV represents a viable mode of action how mAb 2c protected the mice from ARN. We proved this hypothesis using a microfluidic chamber system. Neurons and epithelial cells were cultured in two separate compartments where the neurons sent axons via connecting microgrooves to the epithelial cells. Neurons were infected with a reporter HSV-1 strain expressing mCherry, and the co-culture was treated with neutralizing antibodies. In contrast to commercial polyclonal human HSV-neutralizing immunoglobulins, mAb 2c effectively blocked the anterograde directed neuron-to-cell transmission of the virus. Our data suggest that the humanized HSV-1/2-gB antibody protects mice from ocular disease by blocking the neuronal spread of HSV. Therefore, mAb hu2c may become a potent novel therapeutic option for severe ocular HSV infections.