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Individuals living in areas where malaria is endemic are repeatedly exposed to many different malaria parasite antigens. Studies on naturally acquired antibody-mediated immunity to clinical malaria have largely focused on the presence of responses to individual antigens and their associations with decreased morbidity. We hypothesized that the breadth (number of important targets to which antibodies were made) and magnitude (antibody level measured in a random serum sample) of the antibody response were important predictors of protection from clinical malaria. We analyzed naturally acquired antibodies to five leading Plasmodium falciparum merozoite-stage vaccine candidate antigens, and schizont extract, in Kenyan children monitored for uncomplicated malaria for 6 months (n ‫؍‬ 119). Serum antibody levels to apical membrane antigen 1 (AMA1) and merozoite surface protein antigens (MSP-1 block 2, MSP-2, and MSP-3) were inversely related to the probability of developing malaria, but levels to MSP-1 19 and erythrocyte binding antigen (EBA-175) were not. The risk of malaria was also inversely associated with increasing breadth of antibody specificities, with none of the children who simultaneously had high antibody levels to five or more antigens experiencing a clinical episode (17/119; 15%; P ‫؍‬ 0.0006). Particular combinations of antibodies (AMA1, MSP-2, and MSP-3) were more strongly predictive of protection than others. The results were validated in a larger, separate case-control study whose end point was malaria severe enough to warrant hospital admission (n ‫؍‬ 387). These findings suggest that under natural exposure, immunity to malaria may result from high titers antibodies to multiple antigenic targets and support the idea of testing combination blood-stage vaccines optimized to induce similar antibody profiles.

Major advances in the study of the molecular biology of RNA viruses have resulted from the ability to generate and manipulate full-length genomic cDNAs of the viral genomes with the subsequent synthesis of infectious RNA for the generation of recombinant viruses. Coronaviruses have the largest RNA virus genomes and, together with genetic instability of some cDNA sequences in Escherichia coli, this has hampered the generation of a reverse-genetics system for this group of viruses. In this report, we describe the assembly of a full-length cDNA from the positive-sense genomic RNA of the avian coronavirus, infectious bronchitis virus (IBV), an important poultry pathogen. The IBV genomic cDNA was assembled immediately downstream of a T7 RNA polymerase promoter by in vitro ligation and cloned directly into the vaccinia virus genome. Infectious IBV RNA was generated in situ after the transfection of restricted recombinant vaccinia virus DNA into primary chick kidney cells previously infected with a recombinant fowlpox virus expressing T7 RNA polymerase. Recombinant IBV, containing two marker mutations, was recovered from the transfected cells. These results describe a reverse-genetics system for studying the molecular biology of IBV and establish a paradigm for generating genetically defined vaccines for IBV.

A recombinant infectious bronchitis virus (IBV), BeauR-M41(S),Avian infectious bronchitis virus (IBV), a member of the Coronaviridae (order Nidovirales, genus Coronavirus), is a highly infectious pathogen of domestic fowl that replicates primarily in the respiratory tract but also in epithelial cells of the gut, kidney, and oviduct (3, 6, 7). Genetically very similar coronaviruses cause disease in turkeys and pheasants (4, 5). Coronaviruses are enveloped viruses that replicate in the cell cytoplasm and contain an unsegmented, single-stranded, positive-sense RNA genome of 27 to 32 kb (11,16,24).All coronavirus lipid envelopes contain at least three membrane proteins: the spike glycoprotein (S), integral membrane protein (M), and small membrane protein (E). The coronavirus S protein is a type I glycoprotein which oligomerizes in the endoplasmic reticulum and is assembled into virion membranes through noncovalent interactions with the membrane protein (12). Following incorporation into coronavirus particles, the S glycoprotein is responsible for binding to the target cell receptor and fusion of the viral and cellular membranes. The S glycoprotein consists of four domains: a signal sequence that is cleaved during synthesis; the ectodomain, which is present on the outside of the virion particle; the transmembrane region responsible for anchoring the S protein into the lipid bilayer of the virion particle; and the cytoplasmic tail. The IBV S glycoprotein (1,162 amino acids) is cleaved into two subunits, S1 (535 amino acids; 90 kDa), comprising the Nterminal half of the S protein, and S2 (627 amino acids; 84 kDa), comprising the C-terminal half of the S protein. The S2 subunit associates noncovalently with the S1 subunit and contains the transmembrane and C-terminal cytoplasmic tail domains. The S1 subunit contains the receptor-binding activity of the S protein (14, 23). The ectodomain region of the S2 subunit contains a fusion peptide-like region (18) and two heptad repeat regions involved in oligomerization of the S protein (10). The shorter heptad repeat is adjacent to the transmembrane region and consists of a leucine zipper motif (1). Modification of the murine hepatitis coronavirus (MHV) S protein leucine zipper motif affected oligomerization and loss of cell-to-cell fusion (17).Targeted recombination has been used for modifying the MHV S gene (9,19,21). Similarly, a recombinant porcine coronavirus transmissible gastroenteritis virus (TGEV) containing the S gene derived from an enteric TGEV within a respiratory TGEV genome was isolated in vivo and was shown to have acquired an enteric tropism (22). In addition, the S glycoprotein ectodomain of MHV has been replaced with the corresponding sequence from the coronavirus feline infectious peritonitis virus, and the resulting virus acquired the ability to infect feline cells with the concomitant loss of ability to infect murine cells in tissue culture (15). These results demonstrated that the S glycoprotein for a group 2 coronavirus (MHV) or a group 1 coronavirus (TGE...