Gennady V. Rezapkin

Evaluation of Immunogenicity and Protective Properties of Inactivated Poliovirus Vaccines: A New Surrogate Method for Predicting Vaccine Efficacy

An assay for the evaluation of protective properties of inactivated poliovirus vaccines (IPVs) in transgenic (Tg) mice susceptible to poliovirus has been developed and optimized for type 2 IPV. This method was used to compare the immunogenicity and protective properties of experimental IPV produced from the attenuated Sabin strain (sIPV) with those of conventional IPV (cIPV) produced from the wild-type (wt) poliovirus MEF-1 strain. Modified enzyme-linked immunosorbent assays (ELISAs) were used to measure immune response in serum and saliva samples from test mice. Tg mice were vaccinated and were challenged either with wt poliovirus or virulent poliovirus derived from the vaccine strain. Compared with cIPV, sIPV induced lower levels of antibodies and did not completely protect mice against challenge with wt virus but did protect mice against challenge with the virulent vaccine-derived strain. This may be due to an 18% nucleotide difference between the MEF-1 and Sabin 2 strains, resulting in 72 amino acid substitutions and leading to antigenic dissimilarity. Immunological properties of both strains, revealed by cross-neutralization tests and ELISAs, confirmed that MEF-1 possesses broader immunogenicity than does Sabin 2. This animal model may be used for the assessment of new IPVs and of combination vaccines containing an IPV component.

Antigenic Evolution of Vaccine-Derived Polioviruses: Changes in Individual Epitopes and Relative Stability of the Overall Immunological Properties

ABSTRACT The Sabin oral poliovirus vaccine (OPV) readily undergoes changes in antigenic sites upon replication in humans. Here, a set of antigenically altered descendants of the three OPV serotypes (76 isolates) was characterized to determine the driving forces behind these changes and their biological implications. The amino acid residues of OPV derivatives that lie within or close to the known antigenic sites exhibited a marked tendency to be replaced by residues characteristic of homotypic wild polioviruses, and these changes may occur very early in OPV evolution. The specific amino acid alterations nicely correlated with serotype-specific changes in the reactivity of certain individual antigenic sites, as revealed by the recently devised monoclonal antibody-based enzyme-linked immunosorbent assay. In comparison to the original vaccine, small changes, if any, in the neutralizing capacity of human or rabbit sera were observed in highly diverged vaccine polioviruses of three serotypes, in spite of strong alterations of certain epitopes. We propose that the common antigenic alterations in evolving OPV strains largely reflect attempts to eliminate fitness-decreasing mutations acquired either during the original selection of the vaccine or already present in the parental strains. Variability of individual epitopes does not appear to be primarily caused by, or lead to, a significant immune evasion, enhancing only slightly, if at all, the capacity of OPV derivatives to overcome immunity in human populations. This study reveals some important patterns of poliovirus evolution and has obvious implications for the rational design of live viral vaccines.

Spread of vaccine-derived poliovirus from a paralytic case in an immunodeficient child: An insight into the natural evolution of oral polio vaccine

ABSTRACT Sabin strains used in the manufacture of oral polio vaccine (OPV) replicate in the human organism and can give rise to vaccine-derived polioviruses. The increased neurovirulence of vaccine derivatives has been known since the beginning of OPV use, but their ability to establish circulation in communities has been recognized only recently during the latest stages of the polio eradication campaign. This important observation called for studies of their emergence and evolution as well as extensive surveillance to determine the scope of this phenomenon. Here, we present the results of a study of vaccine-derived isolates from an immunocompromised poliomyelitis patient, the contacts, and the local sewage. All isolates were identified as closely related and slightly evolved vaccine derivatives with a recombinant type 2/type 1 genome. The strains also shared several amino acid substitutions including a mutation in the VP1 protein that was previously shown to be associated with the loss of attenuation. Another mutation in the VP3 protein resulted in altered immunological properties of the isolates, possibly facilitating virus spread in immunized populations. The patterns and rates of the accumulation of synonymous mutations in isolates collected from the patient over the extended period of excretion suggest either a substantially nonuniform rate of mutagenesis throughout the genome, or, more likely, the strains may have been intratypic recombinants between coevolving derivatives with different degrees of divergence from the vaccine parent. This study provides insight into the early stages of the establishment of circulation by runaway vaccine strains.

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.

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.

Reevaluation of nucleotide sequences of wild-type and attenuated polioviruses of type 3

Published sequences of wild-type and attenuated Sabin strains of type 3 poliovirus (Leon/37 and Leon 12a(1)b) were derived from cDNA clones. Recent direct sequencing of Sabin 3 RNA showed that it differed from the published sequence in at least two sites. Here results of direct sequencing of genomes of three independently re-derived sub-strains of attenuated Sabin 3 poliovirus used for oral poliovirus vaccine (OPV) production in addition to the most widely used Pfizer sub-strain are reported. The results showed that all four sub-strains of attenuated type 3 poliovirus contain unique patterns of mutations. Two stocks of the wild-type progenitor Leon/37 strain were also sequenced. Analysis of the two samples of Leon/37 virus showed that one of them is much closer to the Sabin 3 strain, and is an intermediate product of the attenuation process. In addition, we created genetically engineered constructs which contained some of the mutations suspected for their possible role in neurovirulence, and tested them in monkeys and in transgenic mice sensitive to poliovirus. The results suggested that none of them increased neurovirulence of the virus, but some may improve virus replication. Therefore the only mutation occurring in Sabin 3 under vaccine production conditions that appears to affect neurovirulence of the virus is the well known U-->C reversion at nucleotide 472.

RECENT ADVANCES IN SAFETY TESTING OF LIVE ORAL POLIOVIRUS VACCINES. • 979

In studies with polioviruses (PV) isolated from infected children, Sabin, seeking suitable strains to attenuate for live oral PV vaccine (OPV), derived substrains with exceptionally low virulence for monkeys inoculated intraspinally-the species and route of inoculation most sensitive to residual neurovirulence. Because OPV strains tend to undergo partial genetic reversion, it is important to verify that live vaccine preparations retain all markers of attenuation, especially lack of neurovirulence. For many years US federal regulations have required that each monovalent virus pool be tested for neurovirulence by injection into the spinal cords of monkeys before incorporation into trivalent OPV. Monkey neurovirulence testing (MNVT) is expensive and uses very large numbers of animals-testing for a single lot of trivalent OPV requiring 88 monkeys or more. We have studied three tests that might eventually reduce the reliance on MNVT if not replace it: 1) A molecular assay to detect increased content in PV vaccine of those virions with revertant mutations at sites in the viral genome thought to be responsible for neurovirulence (“mutant analysis by polymerase chain reaction and restriction enzyme cleavage”/MAPREC). 2) Intraspinal inoculation of transgenic (Tg) mice expressing the human PV-receptor gene and susceptible to infection with PV. 3) Increased replication of neurovirulent strains of type-3 PV in cultures of interferon-treated human neuroblastoma cells. MAPREC and Tg-mouse tests successfully detected preparations of type-3 OPV (the least stable type of OPV most often incriminated in vaccine-associated paralytic poliomyelitis) that had failed MNVT. The neuroblastoma test recently showed promise in detecting revertant type-3 OPV. Both MAPREC and Tg-mouse tests are currently subjects of multi-center studies coordinated through the World Health Organization.

Molecular consistency monitoring of live viral vaccines |[diams]| 823

The replication of viral RNA is error-prone, resulting in a high degree of genetic diversity within natural populations of viruses. Live viral vaccines may undergo significant genetic changes during passage in vitro and after replication in the bodies of vaccinees, often resulting in some loss of attenuation. A classic example of the plasticity of viral RNA genomes can be seen with Sabins attenuated strains of Oral Poliovirus Vaccine (OPV), which partially revert to neurovirulence for monkeys when serially passaged in cell cultures-especially at elevated temperatures and in the intestinal tracts of vaccine recipients. This genetic instability dictates that production conditions for OPV must be strictly monitored to assure that the vaccine maintains the attenuating phenotype. Thus every batch of OPV is required to undergo rigorous consistency testing in monkeys. Recently we demonstrated that quantification of the amount of a neurovirulent mutation in batches of type-3 OPV accurately predicted the results of the monkey neurovirulence test (MNVT), and that this can be used to monitor consistency of type-3 OPV production. In this communication we summarize recent efforts to predict the neurovirulence of type-1 and type-2 OPV by quantifying selected mutations. Monitoring of two mutations in complementary nucleotides 480 and 525 of the 5′-non coding region of type-1 OPV detected breaks in consistency of vaccine production with much greater sensitivity than did the MNVT. We also demonstrated that the attenuating phenotype of type-1 OPV is much more stable than those of the other two types of OPV. This higher stability results from the relatively weak contribution of 480-G->A and 525-U >C mutations to neurovirulence, and the virtual absence of any other genetically unstable sites. For type-2 OPV, an A->G mutation at nucleotide 481 plays a major role in reversion to virulence, but its virulence is modulated by the presence of two additional mutations (3363-A->G and 3364-G->A) in the gene coding the VP1 capsid protein. However, genetically engineered viral constructs containing these newly identified mutations without 481-A >G reversion had only low levels of neurovirulence, suggesting that they play only an accessory role in the neurovirulence of type-2 poliovirus vaccine strains. We propose that the monitoring of molecular consistency of other live attenuated viral vaccines quantifying both mutant nucleotides of known virulence as well as other unstable nucleotides that tend to accumulate during production may provide a useful test to control vaccine quality.