Immunological Aspects of Circulating DNA

BackgroundIn October 2007, the working group CEN/TC 216 of the European Committee for standardisation suggested that the Sabin oral poliovirus vaccine type 1 strain (LSc-2ab) presently used for virucidal tests should be replaced by another attenuated vaccine poliovirus type 1 strain, CHAT. Both strains were historically used as oral vaccines, but the Sabin type 1 strain was acknowledged to be more attenuated. In Germany, vaccination against poliomyelitis was introduced in 1962 using the oral polio vaccine (OPV) containing Sabin strain LSc-2ab. The vaccination schedule was changed from OPV to an inactivated polio vaccine (IPV) containing wild polio virus type 1 strain Mahoney in 1998. In the present study, we assessed potential differences in neutralising antibody titres to Sabin and CHAT in persons with a history of either OPV, IPV, or OPV with IPV booster.MethodsNeutralisation poliovirus antibodies against CHAT and Sabin 1 were measured in sera of 41 adults vaccinated with OPV. Additionally, sera from 28 children less than 10 years of age and immunised with IPV only were analysed. The neutralisation assay against poliovirus was performed according to WHO guidelines.ResultsThe neutralisation activity against CHAT in adults with OPV vaccination history was significantly lower than against Sabin poliovirus type 1 strains (Wilcoxon signed-rank test P < 0.025). In eight sera, the antibody titres measured against CHAT were less than 8, although the titre against Sabin 1 varied between 8 and 64. Following IPV booster, anti-CHAT antibodies increased rapidly in sera of CHAT-negative adults with OPV history. Sera from children with IPV history neutralised CHAT and Sabin 1 strains equally.ConclusionThe lack of neutralising antibodies against the CHAT strain in persons vaccinated with OPV might be associated with an increased risk of reinfection with the CHAT polio virus type 1, and this implies a putative risk of transmission of the virus to polio-free communities. We strongly suggest that laboratory workers who were immunised with OPV receive a booster vaccination with IPV before handling CHAT in the laboratory.

Meignier et al. (1986) report the results of exposure of C57BL/6NCr mice to vaginal plugs containing live or inactivated herpes simplex virus 1 or 2 (HSV-1 or HSV-2) or recombinant viruses 5 times a week for up to 114 weeks. Genital organs showing abnormalities were transplanted into nude mice. Of 33 transplants, 13 produced subcutaneous tumors in nude mice and 12 were subsequently transplanted into C57BL/6NCr mice. We report that the DNA extracted from coded tumor tissues of nude mice and from normal viscera of the same rodents did not hybridize with HSV-1 and HSV-2 DNA probes representing the viral genomic regions shown previously to be capable of morphologically transforming cells in culture. The sensitivity of the assays was such that we could detect 0.5 copies of the HSV sequences of complexity equal to or greater than 1 Kbp per cell DNA equivalent. To control for the sensitivity of the assays in the actual hybridizations, the tumor-cell DNA was also hybridized with a beta-globin mouse DNA probe. A striking feature of these control hybridizations was the detection of beta-globin polymorphism in some nude mouse tumors. The beta-globin polymorphism allowed us to conclude that the analyzed tissues contained significant amounts of the tumor cells occurring in the C57BL/6NCr mice.

Please cite this paper as: He et al. (2012) Mucosal antibody responses are directed by viral burden in children with acute influenza infection. Influenza and Other Respiratory Viruses DOI: 10.1111/j.1750‐2659.2012.00346.x.Background Influenza infection causes excess hospitalizations and deaths in younger patients, but susceptibility to severe disease is poorly understood. While mucosal antibodies can limit influenza‐associated infection and disease, little is known about acute mucosal antibody responses to influenza infection.Objectives These studies characterize mucosal antiviral antibody production in children during lower respiratory infection (LRI) with H1N1 influenza versus other viral LRI and examine the relationship between mucosal antiviral antibodies and protection against severe disease.Methods B lymphocytes were assessed by immunohistochemistry in lung tissue from infants with fatal acute seasonal influenza infection. Nasopharyngeal secretions (NPS) were obtained at presentation from children with acute respiratory illness, including H1N1 (2009) influenza infection. Total and antiviral antibodies, and inflammatory and immune mediators, were quantified by ELISA. Neutralizing activity in NPS was detected using a pseudotyped virus assay. Viral burden was assessed by qPCR.Results and conclusions B lymphocytes were abundant in lung tissue of infants with fatal acute influenza LRI. Among surviving children with H1N1 infection, only a small subset (11%) demonstrated H1N1 neutralizing activity in NPS. H1N1 neutralizing activity coincided with high local levels of antiviral IgM, IgG and IgA, greater detection of inflammatory mediators, and higher viral burden (P = 0·016). Patients with mucosal antiviral antibody responses demonstrated more severe respiratory symptoms including greater hypoxia (P = 0·0018) and pneumonia (P = 0·038). These patients also trended toward younger age, longer duration of illness and longer hospital stays. Prophylaxis strategies that heighten neutralizing antibody production in the mucosa are likely to benefit both older and younger children.

Fibroblast growth factor 8b (FGF8b) has been implicated in oncogenesis of sex hormone-related malignancies. A murine monoclonal anti-FGF8 antibody, KM1334, has been raised against a FGF8b-derived peptide and shown to neutralize FGF8b activity in an androgen-dependent mouse mammary cell line (SC-3) in vitro growth. The purpose of this study was to evaluate KM1334 as a therapeutic agent for FGF8-dependent cancer. Specificity and neutralizing activity of KM1334 were examined in vitro. In vivo therapeutic studies were done in nude mice bearing SC-3 tumors s.c. KM1334 recognized FGF8b and FGF8f specifically out of four human FGF8 isoforms and showed little binding to other members of FGF family. Neutralizing activity of KM1334 was confirmed by both blocking of FGF8b binding to its three receptors (FGFR2IIIc, FGFR3IIIc, and FGFR4) and FGF8b-induced phosphorylation of FGFR substrate 2alpha and extracellular signal-regulated kinase 1/2 in SC-3 cells. The in vitro inhibitory effect could be extended to in vivo tumor models, where KM1334 caused rapid regression of established SC-3 tumors in nude mice. This rapid regression of tumors after KM1334 treatment was explained by two independent mechanisms: (a) decreased DNA synthesis, as evidenced by a decrease in uptake of 5-bromo-2'-deoxyuridine, and (b) induction of apoptosis as shown by the terminal deoxynucleotidyl transferase-mediated nick end labeling assay. KM1334 possesses strong blocking activity in vitro and antitumor activity in vivo and therefore may be an effective therapeutic candidate for the treatment of cancers that are dependent on FGF8b signaling for growth and survival.

Zika virus (ZIKV) emerged as an important infectious disease agent in Brazil in 2016. Infection usually leads to mild symptoms, but severe congenital neurological disorders and Guillain-Barré syndrome have been reported following ZIKV exposure. Creating an effective vaccine against ZIKV is a public health priority. We describe the protective effect of an already licensed attenuated yellow fever vaccine (YFV, 17DD) in type-I interferon receptor knockout mice (A129) and immunocompetent BALB/c and SV-129 (A129 background) mice infected with ZIKV. YFV vaccination provided protection against ZIKV, with decreased mortality in A129 mice, a reduction in the cerebral viral load in all mice, and weight loss prevention in BALB/c mice. The A129 mice that were challenged two and three weeks after the first dose of the vaccine were fully protected, whereas partial protection was observed five weeks after vaccination. In all cases, the YFV vaccine provoked a substantial decrease in the cerebral viral load. YFV immunization also prevented hippocampal synapse loss and microgliosis in ZIKV-infected mice. Our vaccine model is T cell-dependent, with AG129 mice being unable to tolerate immunization (vaccination is lethal in this mouse model), indicating the importance of IFN-γ in immunogenicity. To confirm the role of T cells, we immunized nude mice that we demonstrated to be very susceptible to infection. Immunization with YFV and challenge 7 days after booster did not protect nude mice in terms of weight loss and showed partial protection in the survival curve. When we evaluated the humoral response, the vaccine elicited significant antibody titers against ZIKV; however, it showed no neutralizing activity in vitro and in vivo. The data indicate that a cell-mediated response promotes protection against cerebral infection, which is crucial to vaccine protection, and it appears to not necessarily require a humoral response. This protective effect can also be attributed to innate factors, but more studies are needed to strengthen this hypothesis. Our findings open the way to using an available and inexpensive vaccine for large-scale immunization in the event of a ZIKV outbreak.

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.

Analysis of a Therapeutic Antibody Cocktail Reveals Determinants for Cooperative and Broad Ebolavirus Neutralization

BackgroundIn principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms - e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV).MethodsBreast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII+-cell depletion) in nude mice.ResultsLate stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII+/CD31+ vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII+-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis.ConclusionsTaken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome.

Immunization with HIV-1 gp41 Subunit Virosomes Induces Mucosal Antibodies Protecting Nonhuman Primates against Vaginal SHIV Challenges

IgG devoid of neutralizing activity and isolated from donor plasma by chromatography formed immune complexes with coxsackievirus B4 (CVB4) and significantly increased the infection of peripheral blood mononuclear cells with CVB4. The major host cells for CVB4 infection enhanced with IgG are monocytic CD14+ cells. The roles of CVB and adenovirus receptor and Fcgamma receptor II and III have been shown. Increased viral replication and the release of infectious particles were demonstrated when interferon (IFN)-alpha produced by infected cells was first neutralized by use of antibodies. The CVB4 IgG-induced synthesis of IFN-alpha by monocytes reflected entry and uncoating of CVB4 but not of viral replication and required the presence of CVB4 RNA inside the cells. Thus, CVB4 can infect monocytes by an antibody-dependent mechanism through interactions between the virus, antiviral antibodies, and specific receptors that result in IFN-alpha production.

Complement Protein C1q Reduces the Stoichiometric Threshold for Antibody-Mediated Neutralization of West Nile Virus

A bispecific nanobody targeting the dimerization interface of epidermal growth factor receptor: Evidence for tumor suppressive actions in vitro and in vivo

Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the antibody repertoire induced after infection or after vaccination against RSV, we investigated antibody levels against fusion (F) protein, attachment (G) protein, and F-specific epitopes in human and animal sera. The results indicate the importance of prefusion-specific antigenic site Ø antibodies as well as of antibodies targeting other epitopes in virus neutralization. However, vaccination of cotton rats with FI-RSV specifically resulted in the induction of weakly neutralizing, antigenic site I-specific antibodies, which may play a role in the enhancement of disease observed after vaccination with such preparations.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against five monoclonal antibodies (MAbs) specific for different antigenic sites on the hemagglutinin (HA) of influenza virus Mem71H-BelN (H3N1) [A/Memphis/1/71 (H3N2) x A/Bel/42 (H1N1)]. Each of the anti-Id sera was directed predominantly towards a unique (private) idiotype of the immunizing MAb, none of the five idiotypes being detectable in pooled BALB/c antisera against Mem71H-BelN virus or on most other anti-HA MAbs tested. Partial idiotypic sharing was observed, however, between certain MAbs, from different mice, having the same or similar epitope specificity for HA. When used as immunogens in BALB/c mice, two of the five anti-Id preparations induced antibodies that reacted with Mem71H-BelN virus and displayed neutralizing activity. Mice of other inbred strains responded similarly, indicating that the response was not genetically restricted by the Igh locus. From their pattern of reactivity with mutants of Mem71H-BelN virus with known single amino acid substitutions in the HA molecule, the antiviral antibodies elicited by anti-Id antibodies were shown to be directed to the same antigenic site on A/Memphis/1/71 HA as the original immunizing MAb (site A or site E, respectively). However, several of these antisera were shown to contain additional distinct subpopulations of antibodies specific for heterologous influenza A virus strains, either of the H3 subtype or of a different HA subtype (H1 or H2). Since the induction of antibodies to HA of different subtypes is not a feature of the antibody response to influenza virus itself, their induction by anti-Id antibodies merits further investigation.

A promising approachto combat Covid-19 infections is the developmentof effective antiviral antibodies that target the SARS-CoV-2 spikeprotein. Understanding the structures and molecular mechanisms underlyingthe binding of antibodies to SARS-CoV-2 can contribute to quicklyachieving this goal. Recently, a cocktail of REGN10987 and REGN10933antibodies was shown to be an excellent candidate for the treatmentof Covid-19. Here, using all-atom steered molecular dynamics and coarse-grainedumbrella sampling, we examine the interactions of the receptor-bindingdomain (RBD) of the SARS-CoV-2 spike protein with REGN10987 and REGN10933separately as well as together. Both computational methods show thatREGN10933 binds to RBD more strongly than REGN10987. Importantly,the cocktail binds to RBD (simultaneous binding) more strongly thanits components. The dissociation constants of REGN10987-RBD and REGN10933-RBDcomplexes calculated from the coarse-grained simulations are in goodagreement with the experimental data. Thus, REGN10933 is probablya better candidate for treating Covid-19 than REGN10987, althoughthe cocktail appears to neutralize the virus more efficiently thanREGN10933 or REGN10987 alone. The association of REGN10987 with RBDis driven by van der Waals interactions, while electrostatic interactionsdominate in the case of REGN10933 and the cocktail. We also studiedthe effectiveness of these antibodies on the two most dangerous variantsDelta and Omicron. Consistent with recent experimental reports, ourresults confirmed that the Omicron variant reduces the neutralizingactivity of REGN10933, REGN10987, and REGN10933+REGN10987 with theK417N, N440K, L484A, and Q498R mutations playing a decisive role,while the Delta variant slightly changes their activity.