Generation and Characterization of aHumanized Monoclonal Antibody for Prophylaxis and Therapy of Herpes Simplex Virus Infections

Herpes Simplex Viren sind weltweit verbreitet und gehören zu den häufigsten viralen Infektionserregern. Reaktivierungen dieser lebenslang im Wirtsorganismus persistierenden Viren sind insbesondere bei Patienten mit einer schweren Immunsuppression mit lebensbedrohlichen Komplikationen assoziiert. Der dadurch notwendige häufige und langandauernde Einsatz etablierter Virustatika führt zu einer zunehmenden Ausbildung resistenter Virusstämme, die eine rasch fortschreitende, generalisierte Infektion mit tödlichem Verlauf zur Folge haben können. Im Rahmen der vorliegenden Arbeit wurde der gegenüber HSV-1 und HSV-2 hochwirksame murine monoklonale Antikörper mAk 2c für eine spätere Anwendung beim Menschen präklinisch evaluiert. Die Untersuchungen der funktionellen Eigenschaften der Antikörperfragmente 2c-F(ab´)2, 2c-Fab und 2c-scFv im direkten Vergleich zum parentalen mAk 2c zeigten, dass die Antikörpervalenz für die antivirale Wirksamkeit des Antikörpers von entscheidender Bedeutung war. Die bivalenten Antikörper mAk 2c und 2c-F(ab´)2 zeichneten sich im Gegensatz zu den monovalenten Antikörper-Fragmenten 2c-Fab und 2c-scFv durch eine extrem effiziente Virusneutralisation aus und konnten den „cell-to-cell spread“ wirksam inhibieren. Im Gegensatz zur Maus werden beim Mensch Antikörper mit dieser Spezifität nicht gebildet. Da murine Antikörper für den Menschen immunogen sind, wurde die Immunogenität des mAk 2c durch Humanisierung nahezu vollständig eliminiert. Die funktionellen Eigenschaften des parentalen mAk 2c konnten dabei vollständig erhalten werden. Bereits die durch alleiniges CDR-Grafting generierte humanisierte Variante mAk hu2c V1 wies eine zum mAk 2c vergleichbare Affinität auf und war zur gleichermaßen effizienten Virusneutralisation sowie Inhibition des „cell-to-cell spreads“ in der Lage. Darüberhinaus konnte die antivirale Wirksamkeit des mAk hu2c V1 an einem breiten Kollektiv von klinischen HSV-1/HSV-2 Isolaten ohne oder mit nachgewiesener Virustatika-Resistenz sowie Laborstämmen demonstriert werden. Sämtliche der untersuchten Isolate wurden durch den humanisierten Antikörper mit derselben Effektivität neutralisiert wie die Laborstämme des entsprechenden Typs. Die Untersuchung des Neutralisationspotentials des mAk hu2c V1 im NOD/SCID-Mausmodell zeigte erstmals, dass die alleinige Antikörpergabe ohne zusätzliche Aktivierung von Immun-Effektorfunktionen einen wirksamen Schutz vor einer sonst tödlich verlaufenden Infektion vermittelt. Abschließend wurde die Fähigkeit des mAk hu2c V1 zur Neutralisation des gegenüber etablierten Virustatika multiresistenten Isolates HSV-1 R10.2 im Tiermodell untersucht. Die Gabe des humanisierten Antikörpers im Zuge einer postexpositionellen Immunprophylaxe führte im Gegensatz zur Behandlung mit Aciclovir zu einer wirksamen Eliminierung der Infektion und zum vollständigen Schutz der immunsupprimierten Versuchstiere vor einem letalen Infektionsverlauf. Die im Rahmen dieser Arbeit erfolgreich durchgeführte Humanisierung des anti-HSV-Antikörpers stellt eine vielversprechende Grundlage für die klinische Weiterentwicklung dieses Antikörpers als eine äußerst wirksame Virustatikum-Alternative zur Prophylaxe und Therapie von Herpes Simplex Infektionen dar.Herpes Simplex Virus type 1 (HSV-1) and type 2 (HSV-2) are widely spread viruses in adults. After primary lytic infection, HSV establishes a lifelong latent infection followed by periodical recurrence. Aciclovir (ACV), Foscarnet and Cidofovir are the drugs of choice for the treatment of acute HSV infections. However, in immunocompromized individuals like HIV/AIDS patients or transplant recipients, increasing occurrence of ACV- and multiresistant HSV is associated with severe life-threatening infections. Human anti-HSV-antibodies generated after primary infection are essential for the establishment of the latent infection, but do not inhibit the virus proliferation or the HSV-typical cell-to-cell spread. Thus, development of novel drugs for overcoming HSV drug resistance is of great importance. In the current work, the murine monoclonal antibody mAb 2c, which proved to be extremely effective in suppression of HSV infections in mice, was evaluated as a novel compound to overcome resistant HSV-infections. Furthermore, it could be demonstrated that antibody valency has a major effect on antiviral efficiency. The bivalent antibodies mAb 2c and F(ab)2 fragments were extremely efficacious in clearance of free virus and inhibition of the cell-to-cell spread, while monovalent 2c-Fab and 2c-scFv fragments induced a 300 fold lower neutralization and were not capable to block viral cell-to-cell spread. Major impediment to the clinical utility of murine antibodies is their potential to elicit human anti-murine antibody response in patients. Thus, the immunogenic potential of mAb 2c was eliminated by a new humanization approach. The creation of the humanized mAk hu2c V1 was accomplished by inserting the appropriate CDR coding segments, responsible for the desired binding properties, into a human antibody "scaffold". The affinity, the virus neutralization and the cell-to-cell spread inhibition results showed that the humanized antibody mAb hu2c V1 retained the biological properties of the parental murine monoclonal antibody. Furthermore, mAb hu2c V1 showed extraordinary neutralization efficiency towards ACV- and multiresistant HSV-1 and HSV-2 strains in vitro. Finally, it could be shown that mAb hu2c V1 was capable to eliminate virus from the vaginal mucosa and to prevent severe immunodeficient NOD/SCID mice from a lethal outcome of HSV-infection. In contrast to ACV, the humanized antibody efficiently neutralized the multiresistant (ACV, PFA, CDV) HSV-1 R-10.2 strain in NOD/SCID mice. Thus, the obtained data hold great promise for the further development of mAb hu2c V1 as an alternative drug for HSV therapy

Large-scale screening of HCMV-seropositive blood donors indicates that HCMV effectively escapes from antibodies by cell-associated spread

Immunoglobulins are only moderately effective for the treatment of human cytomegalovirus (HCMV) infections, possibly due to ineffectiveness against cell-associated virus spread. To overcome this limitation, we aimed to identify individuals with exceptional antibodies in their plasma that can efficiently block the cell-associated spread of HCMV. A Gaussia luciferase-secreting mutant of the cell-associated HCMV strain Merlin was generated, and luciferase activity evaluated as a readout for the extent of cell-associated focal spread. This reporter virus-based assay was then applied to screen plasma samples from 8400 HCMV-seropositive individuals for their inhibitory effect, including direct-acting antiviral drugs as positive controls. None of the plasmas reduced virus spread to the level of these controls. Even the top-scoring samples that partially reduced luciferase activity in the screening assay failed to inhibit focal growth when reevaluated with a more accurate, immunofluorescence-based assay. Selected sera with high neutralizing capacity against free viruses were analyzed separately, and none of them prevented the focal spread of three recent clinical HCMV isolates nor reduced the number of particles transmitted, as demonstrated with a fluorescent Merlin mutant. We concluded that donors with cell-to-cell-spread-inhibiting plasma are nonexistent or extremely rare, emphasizing cell-associated spread as a highly efficient immune escape mechanism of HCM

Low avidity circulating SARS-CoV-2 reactive CD8+ T cells with proinflammatory TEMRA phenotype are associated with post-acute sequelae of COVID-19

The role of adaptive SARS-CoV-2 specific immunity in post-acute sequelae of COVID-19 (PASC) is not well explored, although a growing population of convalescent COVID-19 patients with manifestation of PASC is observed. We analyzed the SARS-CoV-2-specific immune response, via pseudovirus neutralizing assay and multiparametric flow cytometry in 40 post-acute sequelae of COVID-19 patients with non-specific PASC manifestation and 15 COVID-19 convalescent healthy donors. Although frequencies of SARS-CoV-2-reactive CD4+ T cells were similar between the studied cohorts, a stronger SARS-CoV-2 reactive CD8+ T cell response, characterized by IFNγ production and predominant TEMRA phenotype but low functional TCR avidity was detected in PASC patients compared to controls. Of interest, high avidity SARS-CoV-2-reactive CD4+ and CD8+ T cells were comparable between the groups demonstrating sufficient cellular antiviral response in PASC. In line with the cellular immunity, neutralizing capacity in PASC patients was not inferior compared to controls. In conclusion, our data suggest that PASC may be driven by an inflammatory response triggered by an expanded population of low avidity SARS-CoV-2 reactive pro-inflammatory CD8+ T cells. These pro-inflammatory T cells with TEMRA phenotype are known to be activated by a low or even without TCR stimulation and lead to a tissue damage. Further studies including animal models are required for a better understanding of underlying immunopathogensis. Summary: A CD8+ driven persistent inflammatory response triggered by SARS-CoV-2 may be responsible for the observed sequelae in PASC patients

Antibody-based immunotherapy of aciclovir resistant ocular herpes simplex virus infections

The increasing incidence of aciclovir- (ACV) resistant strains in patients with ocular herpes simplex virus (HSV) infections is a major health problem in industrialized countries. In the present study, the humanized monoclonal antibody (mAb) hu2c targeting the HSV-1/2 glycoprotein B was examined for its efficacy towards ACV-resistant infections of the eye in the mouse model of acute retinal necrosis (ARN). BALB/c mice were infected by microinjection of an ACV-resistant clinical isolate into the anterior eye chamber to induce ARN and systemically treated with mAb hu2c at 24 h prior (pre-e

Role of L-Particles during Herpes Simplex Virus Infection

Infection of eukaryotic cells with α-herpesviruses results in the formation and secretion of infectious heavy particles (virions; H-particles) and non-infectious light particles (L-particles). Herpes simplex virus type 1 (HSV-1) H-particles consist of a genome-containing capsid surrounded by tegument proteins and a glycoprotein-rich lipid bilayer. Non-infectious L-particles are composed mainly of envelope and tegument proteins and are devoid of capsids and viral DNA. L-particles were first described in the early nineties and from then on investigated for their formation and role during virus infection. The development and secretion of L-particles occur simultaneously to the assembly of complete viral particles. HSV-1 L-particles are assembled by budding of condensed tegument into Golgi-delivered vesicles and are capable of delivering their functional content to non-infected cells. Thereby, HSV-1 L-particles contribute to viral pathogenesis within the infected host by enhancing virion infectivity and providing immune evasion functions. In this review we discuss the emergence of HSV-1 L-particles during virus replication and their biological functions described thus far

Rapid Quantification of SARS-CoV-2-Neutralizing Antibodies Using Propagation-Defective Vesicular Stomatitis Virus Pseudotypes.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2, a new member of the genus Betacoronavirus, is a pandemic virus, which has caused numerous fatalities, particularly in the elderly and persons with underlying morbidities. At present, there are no approved vaccines nor antiviral therapies available. The detection and quantification of SARS-CoV-2-neutralizing antibodies plays a crucial role in the assessment of the immune status of convalescent COVID-19 patients, evaluation of recombinant therapeutic antibodies, and the evaluation of novel vaccines. To detect SARS-CoV-2-neutralizing antibodies, classically, a virus-neutralization test has to be performed at biosafety level 3, considerably limiting the general use of this test. In the present work, a biosafety level 1 pseudotype virus assay based on a propagation-incompetent vesicular stomatitis virus (VSV) has been used to determine the neutralizing antibody titers in convalescent COVID-19 patients. The neutralization titers in serum of two independently analyzed patient cohorts were available within 18 h and correlated well with those obtained with a classical SARS-CoV-2 neutralization test (Pearson correlation coefficients of r = 0.929 and r = 0.939, respectively). Most convalescent COVID-19 patients had only low titers of neutralizing antibodies (ND50 < 320). The sera of convalescent COVID-19 patients also neutralized pseudotype virus displaying the SARS-CoV-1 spike protein on their surface, which is homologous to the SARS-CoV-2 spike protein. In summary, we report a robust virus-neutralization assay, which can be used at low biosafety level 1 to rapidly quantify SARS-CoV-2-neutralizing antibodies in convalescent COVID-19 patients and vaccinated individuals

Cellular and Humoral Immunity after the Third Vaccination against SARS-CoV-2 in Hematopoietic Stem-Cell Transplant Recipients

Protecting vulnerable groups from severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection is mandatory. Immune responses after a third vaccination against SARS-CoV-2 are insufficiently studied in patients after hematopoietic stem-cell transplantation (HSCT). We analyzed immune responses before and after a third vaccination in HSCT patients and healthy controls. Cellular immunity was assessed using interferon-gamma (IFN-&gamma;) and interleukin-2 (IL-2) ELISpots. Furthermore, this is the first report on neutralizing antibodies against 11 variants of SARS-CoV-2, analyzed by competitive fluorescence assay. Humoral immunity was also measured by neutralization tests assessing cytopathic effects and by ELISA. Neither HSCT patients nor healthy controls displayed significantly higher SARS-CoV-2-specific IFN-&gamma; or IL-2 responses after the third vaccination. However, after the third vaccination, cellular responses were 2.6-fold higher for IFN-&gamma; and 3.2-fold higher for IL-2 in healthy subjects compared with HSCT patients. After the third vaccination, neutralizing antibodies were significantly higher (p &lt; 0.01) in healthy controls, but not in HSCT patients. Healthy controls vs. HSCT patients had 1.5-fold higher concentrations of neutralizing antibodies against variants and 1.2-fold higher antibody concentrations against wildtype. However, half of the HSCT patients exhibited neutralizing antibodies to variants of SARS-CoV-2, which increased only slightly after a third vaccination

Antiviral Active Compounds Derived from Natural Sources against Herpes Simplex Viruses

Herpes simplex viruses (HSV) are ubiquitously distributed with a seroprevalence ranging up to 95% in the adult population. Refractory viral infections with herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) represent a major global health issue. In particular, the increasing occurrence of resistance to conventional antiviral drugs make the therapy of such infections even more challenging. For instance, the frequent and long-term use of acyclovir and other nucleoside analogues targeting the viral DNA-polymerase enhance the development of resistant viruses. Particularly, the incidental increase of those strains in immunocompromised patients is alarming and represent a major health concern. Alternative treatment concepts are clearly needed. Natural products such as herbal medicines showed antiherpetic activity in vitro and in vivo and proved to be an excellent source for the discovery and isolation of novel antivirals. By this means, numerous plant-derived compounds with antiviral or antimicrobial activity could be isolated. Natural medicines and their ingredients are well-tolerated and could be a good alternative for treating herpes simplex virus infections. This review provides an overview of the recent status of natural sources such as plants, bacteria, fungi, and their ingredients with antiviral activity against herpes simplex viruses. Furthermore, we highlight the most potent herbal medicines and ingredients as promising candidates for clinical investigation and give an overview about the most important drug classes along with their potential antiviral mechanisms. The content of this review is based on articles that were published between 1996 and 2021

Prospective, Longitudinal Study on Specific Cellular Immune Responses after Vaccination with an Adjuvanted, Recombinant Zoster Vaccine in Kidney Transplant Recipients

Solid organ transplant recipients have an up to ninefold higher risk of varicella&ndash;zoster virus (VZV) reactivation than the general population. Due to lifelong immunosuppressive therapy, vaccination against VZV may be less effective in kidney transplant (KTX) recipients. In the current study, twelve female and 17 male KTX recipients were vaccinated twice with the adjuvanted, recombinant zoster vaccine Shingrix&trade;, which contains the VZV glycoprotein E (gE). Cellular immunity against various VZV antigens was analyzed with interferon-gamma ELISpot. We observed the strongest vaccination-induced changes after stimulation with a gE peptide pool. One month after the second vaccination, median responses were 8.0-fold higher than the responses prior to vaccination (p = 0.0006) and 4.8-fold higher than responses after the first vaccination (p = 0.0007). After the second vaccination, we observed an at least twofold increase in ELISpot responses towards gE peptides in 22 out of 29 patients (76%). Male sex, good kidney function, early time point after transplantation, and treatment with tacrolimus or mycophenolate were correlated significantly with higher VZV-specific cellular immunity, whereas diabetes mellitus was correlated with impaired responses. Thus, our data indicate that vaccination with Shingrix&trade; significantly augmented cellular, VZV gE-specific immunity in KTX recipients, which was dependent on several covariates

HSV-1 Modulates IL-6 Receptor Expression on Human Dendritic Cells

Dendritic cells (DCs) are the guardians of the immune system since they are located in the majority of peripheral tissues. In addition, they are crucial for the induction of an effective immune response based on their unique capacity to stimulate naive T cells. During co-evolution, the human pathogen herpes simplex virus type 1 (HSV-1) has evolved several immune evasion mechanisms in order to subvert the host's immune system especially by targeting DC biology and function. Here we demonstrate that HSV-1 infection influences the IL-6 receptor (IL6R) expression both on protein and mRNA levels in/on human monocyte-derived mature DCs (mDCs). Surprisingly, reduced IL6R expression levels were also observed on uninfected bystander mDCs. Mechanistically, we clearly show that HSV-1-derived non-infectious light (L-) particles are sufficient to trigger IL6R regulation on uninfected bystander mDCs. These L-particles lack the viral DNA-loaded capsid and are predominantly produced during infection of mDCs. Our results show that the deletion of the HSV-1 tegument protein vhs partially rescued the reduced IL6R surface expression levels on/in bystander mDCs. Using a neutralizing antibody, which perturbs the transfer of L-particles to bystander mDCs, was sufficient to rescue the modulation of IL6R surface expression on uninfected bystander mDCs. This study provides evidence that L-particles transfer specific viral proteins to uninfected bystander mDCs, thereby negatively interfering with their IL6R expression levels, however, to a lesser extend compared to H-particles. Due to their immune-modulatory capacity, L-particles represent an elaborated approach of HSV-1-mediated immune evasion