Diana Kouiavskaia

Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor

Significance This study demonstrated that cross-neutralizing anti-poliovirus antibodies bind the site on poliovirus capsid surface that significantly overlaps the binding site of the cellular receptor. A second antibody with similar specificity was isolated by sequential phage display panning, suggesting that cross-reactive anti-poliovirus antibodies may be more prevalent in primates than previously recognized. Binding to the receptor recognition site explains unusually broad specificity of the antibodies. The antibodies bind type 1 and type 2 polioviruses at a slightly different angle, indicating that molecular details of virus–antibody interaction are different and suggesting that further screening or engineering may produce an antibody neutralizing all three serotypes of poliovirus. These results may be used for developing new antiviral strategies for the polio eradication campaign. Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus.

Chimpanzee-Human Monoclonal Antibodies for Treatment of Chronic Poliovirus Excretors and Emergency Postexposure Prophylaxis

ABSTRACT Six poliovirus-neutralizing Fabs were recovered from a combinatorial Fab phage display library constructed from bone marrow-derived lymphocytes of immunized chimpanzees. The chimeric chimpanzee-human full-length IgGs (hereinafter called monoclonal antibodies [MAbs]) were generated by combining a chimpanzee IgG light chain and a variable domain of heavy chain with a human constant Fc region. The six MAbs neutralized vaccine strains and virulent strains of poliovirus. Five MAbs were serotype specific, while one MAb cross-neutralized serotypes 1 and 2. Epitope mapping performed by selecting and sequencing antibody-resistant viral variants indicated that the cross-neutralizing MAb bound between antigenic sites 1 and 2, thereby covering the canyon region containing the receptor-binding site. Another serotype 1-specific MAb recognized a region located between antigenic sites 2 and 3 that included parts of capsid proteins VP1 and VP3. Both serotype 2-specific antibodies recognized antigenic site 1. No escape mutants to serotype 3-specific MAbs could be generated. The administration of a serotype 1-specific MAb to transgenic mice susceptible to poliovirus at a dose of 5 μg/mouse completely protected them from paralysis after challenge with a lethal dose of wild-type poliovirus. Moreover, MAb injection 6 or 12 h after virus infection provided significant protection. The MAbs described here could be tested in clinical trials to determine whether they might be useful for treatment of immunocompromised chronic virus excretors and for emergency protection of contacts of a paralytic poliomyelitis case.

Comparison of the Immunogenicity of Various Booster Doses of Inactivated Polio Vaccine Delivered Intradermally Versus Intramuscularly to HIV-Infected Adults

BACKGROUND Inactivated polio vaccine (IPV) is necessary for global polio eradication because oral polio vaccine can rarely cause poliomyelitis as it mutates and may fail to provide adequate immunity in immunocompromised populations. However, IPV is unaffordable for many developing countries. Intradermal IPV shows promise as a means to decrease the effective dose and cost of IPV, but prior studies, all using 20% of the standard dose used in intramuscular IPV, resulted in inferior antibody titers. METHODS We randomly assigned 231 adults with well-controlled human immunodeficiency virus infection at a ratio of 2:2:2:1 to receive 40% of the standard dose of IPV intradermally, 20% of the standard dose intradermally, the full standard dose intramuscularly, or 40% of the standard dose intramuscularly. Intradermal vaccination was done using the NanoPass MicronJet600 microneedle device. RESULTS Baseline immunity was 87%, 90%, and 66% against poliovirus serotypes 1, 2, and 3, respectively. After vaccination, antibody titers increased a median of 64-fold. Vaccine response to 40% of the standard dose administered intradermally was comparable to that of the standard dose of IPV administered intramuscularly and resulted in higher (although not significantly) antibody titers. Intradermal administration had higher a incidence of local side effects (redness and itching) but a similar incidence of systemic side effects and was preferred by study participants over intramuscular administration. CONCLUSIONS A 60% reduction in the standard IPV dose without reduction in antibody titers is possible through intradermal administration.

New Small-Molecule Inhibitors Effectively Blocking Picornavirus Replication

ABSTRACT Few drugs targeting picornaviruses are available, making the discovery of antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, coxsackievirus B3, and encephalomyocarditis virus. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus life cycle. A4(1) inhibited both the formation and functioning of the replication complexes, while E5(1) and E7(2) were most effective during the formation but not the functioning step. Neither of the compounds significantly inhibited VPg uridylylation. Poliovirus resistant to E7(2) had a G5318A mutation in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds, which target a phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ)-dependent step in viral replication. Analysis of host protein recruitment showed that E7(2) reduced the amount of GBF1 on the replication complexes; however, the level of PI4KIIIβ remained intact. E7(2) as well as another enviroxime-like compound, GW5074, interfered with viral polyprotein processing affecting both 3C- and 2A-dependent cleavages, and the resistant G5318A mutation partially rescued this defect. Moreover, E7(2) induced abnormal recruitment to membranes of the viral proteins; thus, enviroxime-like compounds likely severely compromise the interaction of the viral polyprotein with membranes. A4(1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Multiple attempts to isolate resistant mutants in the presence of A4(1) or E5(1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds. IMPORTANCE Diverse picornaviruses can trigger multiple human maladies, yet currently, only hepatitis A virus and poliovirus can be controlled with vaccination. The development of antipicornavirus therapeutics is also facing significant difficulties because these viruses readily generate resistance to compounds targeting either viral or cellular factors. Here, we describe three novel compounds that effectively block replication of distantly related picornaviruses with minimal toxicity to cells. The compounds prevent viral RNA replication after the synthesis of the uridylylated VPg primer. Importantly, two of the inhibitors are strongly refractory to the emergence of resistant mutants, making them promising candidates for further broad-spectrum therapeutic development. Evaluation of one of the compounds in an in vivo model of poliomyelitis demonstrated partial protection from the onset of paralysis.

Immunologic response to oral polio vaccine in human immunodeficiency virus-infected and uninfected Zimbabwean children.

Background: Poliovirus eradication is dependent on maintaining adequate community-wide levels of serologic protection. Many African countries with conditions that favor continued wild poliovirus propagation also have a high prevalence of pediatric human immunodeficiency virus (HIV) infection. Data are limited regarding the degree of serologic immunity conferred on HIV-infected children after immunization with oral polio vaccine (OPV). Methods: This was a cross-sectional study correlating HIV infection and neutralizing antibodies against poliovirus serotypes 1, 2, and 3 in 95 Zimbabwean children 2 months to 2 years of age, born to HIV-infected mothers, who received OPV according to the national schedule. Results: HIV-infected children had significantly lower rates of seroconversion to all 3 poliovirus serotypes than HIV-uninfected children (60%, 67%, and 47% vs. 96%, 100%, and 82%, P = 0.001, 0.0003, and 0.015 for serotypes 1, 2, and 3 in HIV-infected and uninfected children, respectively, after ≥3 OPV doses). Among poliovirus seroconverters, HIV-infected children also had significantly lower geometric mean titers against serotypes 1 and 2 than HIV-uninfected children (geometric mean titers: 198 and 317 vs. 1193 and 1056, P = 0.032 and 0.050, for serotypes 1 and 2, respectively, after ≥3 OPV doses). In addition, HIV-infected children had significantly higher levels of total IgG and significantly lower CD4% and mean weight than HIV-uninfected children. Of note, none of the HIV-infected children were receiving antiretroviral therapy, and 71% had a CD4% indicating severe immunodeficiency. Conclusions: Pediatric HIV infection is associated with a poor serologic response to OPV, which could pose an obstacle to global polio eradication.

Vaccine Poliovirus Shedding and Immune Response to Oral Polio Vaccine in HIV-Infected and Uninfected Zimbabwean Infants

BACKGROUND With prolonged replication, attenuated polioviruses used in oral polio vaccine (OPV) can mutate into vaccine-derived poliovirus (VDPV) and cause poliomyelitis outbreaks. Individuals with primary humoral immunodeficiencies can become chronically infected with vaccine poliovirus, allowing it to mutate into immunodeficiency-associated VDPV (iVDPV). It is unclear if children perinatally infected with the human immunodeficiency virus (HIV), who have humoral as well as cellular immunodeficiencies, might be sources of iVDPV. METHODS We conducted a prospective study collecting stool and blood samples at multiple time points from Zimbabwean infants receiving OPV according to the national schedule. Nucleic acid extracted from stool was analyzed by real-time polymerase chain reaction for OPV serotypes. RESULTS We analyzed 825 stool samples: 285 samples from 92 HIV-infected children and 540 from 251 HIV-uninfected children. Poliovirus shedding was similar after 0-2 OPV doses but significantly higher in the HIV-infected versus uninfected children after ≥ 3 OPV doses, particularly within 42 days of an OPV dose, independent of seroconversion status. HIV infection was not associated with prolonged or persistent poliovirus shedding. HIV infection was associated with significantly lower polio seroconversion rates. CONCLUSIONS HIV infection is associated with decreased mucosal and humoral immune responses to OPV but not the prolonged viral shedding required to form iVDPV.

Human monoclonal antibodies that neutralize vaccine and wild-type poliovirus strains

An essential requirement for eradication of poliomyelitis is the elimination of circulating vaccine derived polioviruses (cVDPV) and polioviruses excreted by chronically infected individuals with immunodeficiencies (iVDPV). As part of a post-eradication risk management strategy, a human monoclonal antibody (mAb) therapeutic could play a role in halting excretion in asymptomatic carriers and could be used, in combination with vaccines and antiviral drugs, to protect polio-exposed individuals. Cross-neutralizing mAbs may be particularly useful, as they would reduce the number of mAbs needed to create a comprehensive PV therapeutic. We cloned a panel of IgG mAbs from OPV-vaccinated, IPV-boosted healthy subjects. Many of the mAbs had potent neutralizing activities against PV wild-type (WT) and Sabin strains, and two of the mAbs, 12F8 and 1E4, were significantly cross-reactive against types 1 and 2 and types 1 and 3, respectively. Mapping the binding epitopes using strains resistant to neutralization (escape mutants) suggested that cross-specific PV binding epitopes may primarily reside within the canyon region, which interacts with the cellular receptor molecule CD155 and the cross-neutralizing chimpanzee/human mAb, A12. Despite their close proximity, the epitopes for the 12F8 and 1E4 mAbs on Sabin 1 were not functionally identical to the A12 epitope. When tested together, 12F8 and 1E4 neutralized a diverse panel of clinically relevant PV strains and did not exhibit interference. Virus mutants resistant to the anti-poliovirus drug V-073 were also neutralized by the mAbs. The 12F8 and 1E4 mAbs may suitable for use as anti-PV therapeutics.

Repertoire of antibodies against type 1 poliovirus in human sera.

A blocking-ELISA procedure was used to quantify antibodies in sera of humans immunized with poliovirus vaccines. Titers determined by this method demonstrated an excellent correlation with the results of neutralization test. Testing of serum potency with a panel of type 1 poliovirus strains altered antigenically was used to evaluate the composition of polyclonal sera with respect to the epitope specificity of constituent antibodies. Paratope profiles of various polyclonal sera determined by this new method differed, depending on the type of vaccine used for immunization. Antibodies induced in response to inactivated poliovirus vaccine (IPV) contained antibodies directed primarily against antigenic site 1, while sera from recipients of the oral poliovirus vaccine (OPV) contained antibodies to site 3. Antibodies to antigenic sites 2 and 4 were minor constituents in both types of sera. Pre-immunization sera had paratope profiles similar to OPV-induced antisera, allowing the discrimination between antibodies induced by IPV and maternal antibodies. The new method may be useful for analyzing results of clinical trials and to compare immunity induced by different poliovirus vaccines.

Intradermal Inactivated Poliovirus Vaccine: A Preclinical Dose-Finding Study

Intradermal delivery of vaccines has been shown to result in dose sparing. We tested the ability of fractional doses of inactivated poliovirus vaccine (IPV) delivered intradermally to induce levels of serum poliovirus-neutralizing antibodies similar to immunization through the intramuscular route. Immunogenicity of fractional doses of IPV was studied by comparing intramuscular and intradermal immunization of Wistar rats using NanoPass MicronJet600 microneedles. Intradermal delivery of partial vaccine doses induced antibodies at titers comparable to those after immunization with full human dose delivered intramuscularly. The results suggest that intradermal delivery of IPV may lead to dose-sparing effect and reduction of the vaccination cost.

A single chimpanzee-human neutralizing monoclonal antibody provides post-exposure protection against type 1 and type 2 polioviruses

BACKGROUND Development of anti-poliovirus therapies to complement vaccination is an urgent priority. A number of antiviral drugs are in development. Recently we have developed human monoclonal antibodies that could be used for treatment of chronically infected individuals and emergency response to potential reappearance of polioviruses after eradication. OBJECTIVE The aim of this study was to characterize neutralizing activity of anti-poliovirus monoclonal antibody A12 against wild type, vaccine-derived, and drug-resistant poliovirus strains, evaluate in vivo pre- and post-exposure protective properties of the antibody against polioviruses of serotypes 1 and 2, and to determine whether it interferes with response to immunization with poliovirus vaccine. STUDY DESIGN Immunogenicity studies were performed in CD1 mice. Poliovirus neutralizing titers were determined in poliovirus microneutralization assay. Poliovirus immunization-challenge experiments were performed in poliovirus-susceptible TgPVR21 mice. RESULTS We show that monoclonal antibody A12 effectively neutralizes in vitro a broad range of type 1 and type 2 wild and vaccine-derived polioviruses, provides effective pre- and post-exposure protection of TgPVR21 mice from challenge with a lethal dose of poliovirus. Treatment of animals with the antibody concurrent with IPV immunization does not prevent immune response to the vaccine. CONCLUSIONS Anti-poliovirus antibody A12 effectively neutralizes a range of wild and VDPV strains and protectstransgenic mice susceptible to poliovirus against lethal challenge upon pre- and post-exposure administration. This suggests that the antibodies could be used in combination with drugs and/or vaccine to improve their efficacy and prevent emergence of resistant variants, and provides a justification for initiating their clinical evaluation.