Sabin strains of poliovirus used in the manufacture of oral poliovirus vaccine (OPV) are prone to genetic variations that occur during growth in cell cultures and the organisms of vaccine recipients. Such derivative viruses often have increased neurovirulence and transmissibility, and in some cases they can reestablish chains of transmission in human populations. Monitoring for vaccine-derived polioviruses is an important part of the worldwide campaign to eradicate poliomyelitis. Analysis of vaccine-derived polioviruses requires, as a first step, their isolation in cell cultures, which takes significant time and may yield viral stocks that are not fully representative of the strains present in the original sample. Here we demonstrate that full-length viral cDNA can be PCR amplified directly from stool samples and immediately subjected to genomic analysis by oligonucleotide microarray hybridization and nucleotide sequencing. Most fecal samples from healthy children who received OPV were found to contain variants of Sabin vaccine viruses. Sequence changes in the 5 untranslated region were common, as were changes in the VP1-coding region, including changes in a major antigenic site. Analysis of stool samples taken from cases of acute flaccid paralysis revealed the presence of mixtures of recombinant polioviruses, in addition to the emergence of new sequence variants. Avoiding the need for cell culture isolation dramatically shortened the time needed for identification and analysis of vaccine-derived polioviruses and could be useful for preliminary screening of clinical samples. The amplified full-length viral cDNA can be archived and used to recover live virus for further virological studies.Live trivalent oral poliovirus vaccine (OPV) prepared from attenuated Sabin strains is highly efficacious, and its worldwide use resulted in eradication of poliomyelitis in the United States (41) and most other countries (37,38). Roughly half of the approximately eight cases reported yearly in the United States involved immunodeficient individuals. Vaccine-derived poliovirus (VDPV) strains isolated from stools of individuals with vaccine-associated paralytic poliomyelitis (VAPP) have been found to contain a number of mutations and exhibit increased neurovirulence. While some of the mutations occurring in VDPV strains restore the sequences present in the wild-type progenitors of the vaccine strains (direct reversions), other mutations either are incidental or are second-site suppressors of attenuated phenotype. All three Sabin strains contain a single base change in the same domain of the 5Ј untranslated region (5Ј-UTR) that contributes to the attenuated phenotype, as indicated by increased neurovirulence in strains where this base change is reversed or altered by a compensating mutation (17, 31). Such Sabin strain variants with reversion in the 5Ј-UTR have been identified in type 3 (5, 17), type 2 (30), and type 1 (9, 24) isolates from patients with VAPP, as well as from healthy vaccine recipients (1,11,16,42). Isolation of suc...

Macrophages are uniquely responsive to bacterial lipopolysaccharide (LPS) for activation of a number of host defense functions and production of bioactive mediators. One potentially important mediator produced by LPS-stimulated macrophages is interferon (IFN-alpha/beta). In contrast to murine observations, we have observed that freshly isolated human monocytes, purified by counter-current centrifugal elutriation, do not produce interferon in response to LPS. This is not due to a lack of response to LPS, as assessed by the induction of other monokines, or to an incapacity for IFN production, since IFN was inducible by poly-I,C treatment of monocytes in the absence of any other exogenous stimulus. However, human monocytes can be primed for the production of IFN in response to LPS if they are cultured in the presence of either granulocyte-macrophage colony stimulating factor (GM-CSF) or interferon-gamma (IFN-gamma). The IFN secreted is of the alpha subtype. Monocytes primed with GM-CSF or IFN-gamma also maintained LPS responses for production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1). M-CSF did not prime monocytes for LPS-induced IFN production, although it did enhance production of TNF-alpha and promoted monocyte survival. Northern analysis indicated that the induction of IFN-alpha by LPS was regulated primarily at the mRNA level. The highly regulated production of IFN-alpha by monocytes/macrophages has important implications for autocrine action of interferons in the activation and differentiation of these cells.

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.

To examine the defect in cellular immunity in patients with acquired immunodeficiency syndrome (AIDS), we studied in vitro lymphocyte proliferation and interferon (IFN) release in response to cytomegalovirus (CMV) antigen and Concanavalin A mitogen in 40 homosexual men with AIDS, 10 homosexual men with chronic lymphadenopathy syndrome, 7 healthy homosexual men, and 18 healthy heterosexual subjects of either sex. CMV serology by an enzyme-linked immunosorbent assay and viral cultures for CMV were performed. Lymphocytes of patients with AIDS showed impaired CMV-specific release of IFN but normal mitogen-induced IFN release. The defect was not attributable to CMV infection per se. Cell proliferation in response to both CMV antigen and mitogen was impaired in patients with AIDS who had opportunistic infections. The defect could not be attributed to CMV viremia. We concluded that impaired release of IFN in response to a viral antigen is characteristic of lymphocytes in patients with AIDS and that this defect is distinct from a defect in mitogenic responsiveness, which coexists predominantly in patients with opportunistic infections.