The current model of measles virus (MV) pathogenesis implies that apical infection of airway epithelial cells precedes systemic spread. An alternative model suggests that primarily infected lymphatic cells carry MV to the basolateral surface of epithelial cells, supporting MV shedding into the airway lumen and contagion. This model predicts that a mutant MV, unable to enter cells through the unidentified epithelial cell receptor (EpR), would remain virulent but not be shed. To test this model, we identified residues of the MV attachment protein sustaining EpR-mediated cell fusion. These nonpolar or uncharged polar residues defined an area located near the binding site of the signaling lymphocytic activation molecule (SLAM), the receptor for MV on lymphatic cells. We then generated an EpR-blind virus maintaining SLAM-dependent cell entry and inoculated rhesus monkeys intranasally. Hosts infected with the selectively EpR-blind MV developed rash and anorexia while averaging slightly lower viremia than hosts infected with wild-type MV but did not shed virus in the airways. The mechanism restricting shedding was characterized using primary well-differentiated human airway epithelial cells. Wild-type MV infected columnar epithelial cells bearing tight junctions only when applied basolaterally, while the EpR-blind virus did not infect these cells. Thus, EpR is probably a basolateral protein, and infection of the airway epithelium is not essential for systemic spread and virulence of MV.

Patients recruited in virus-based cancer clinical trials and immunocompromised individuals in need of vaccination would profit from viral strains with defined attenuation mechanisms. We generated measles virus (MV) strains defective for the expression of either the V protein, a modulator of the innate immune response, or the C protein, which has multiple functions. The virulence of these strains was compared with that of the parental wild-type MV in a natural host, Macaca mulatta. Skin rash, viremia, and the strength of the innate and adaptive immune responses were characterized in groups of six animals. Replication of V-or C-proteindefective viruses was short-lived and reached lower levels in peripheral blood mononuclear cells and lymphatic organs compared to the wild-type virus; none of the mutants reverted to the wild type. The neutralizing antibody titers and MV-specific T-cell responses were equivalent in monkeys infected with the viral strains tested, documenting strong adaptive immune responses. In contrast, the inflammatory response was better controlled by wild-type MV, as revealed by inhibition of interleukin-6 and tumor necrosis factor alpha transcription. The interferon response was also better controlled by the wild-type virus than by the defective viruses. Since V-and C-defective MVs induce strong adaptive immune responses while spreading less efficiently, they may be developed as vaccines for immunocompromised individuals. Moreover, MV unable to interact with single innate immunity proteins may be developed for preferential replication in tumors with specific contexts of vulnerability.Certain safe and effective live attenuated vaccines were generated simply by repeated passage of virulent strains on heterologous cells, resulting in multiple mutations and sometimes gene deletions (30,43). Nevertheless, vaccine strains with defined mechanisms and levels of attenuation would be preferable for new applications of viruses for cancer therapy (18,22) and the vaccination of immunocompromised hosts (37). Many viruses express proteins that control the antiviral response, and viruses without these proteins are promising candidates for specific vector and vaccine applications. Measles virus (MV) host control proteins include V protein, whose mechanisms of innate immunity modulation are well understood, and C protein, whose mechanisms of action are less well defined. Both V and C proteins are dispensable for replication in cultivated cells (35,41).The V protein shares the amino-terminal domain of the P protein, but its 68 carboxyl-terminal highly conserved amino acids forming a zinc-binding domain (31) are translated from a different open reading frame (ORF) accessed by the cotranscriptional insertion of a pseudotemplate G residue (9). MV V functions as an interferon (IFN) antagonist through its interaction with both MDA5 and the STAT proteins (3,7,8,10,27,29,47). V can also interfere with signaling by interleukin-6 (IL-6) (29). Interference with the IFN response facilitates virus replication by blocking the i...

Vaccines based on live viruses can be highly effective and easy to produce and deliver. Smallpox was eradicated with live vaccinia virus, and a live attenuated poliovirus vaccine has been at the core of the polio eradication campaign (17). Worldwide measles virus (MV) vaccination prevents an estimated 80 million cases and 4.5 million deaths annually (45) with minimal severe adverse effects, on average less than 10 in 1 million doses (34). With the currently recommended vaccination scheme, the first dose of vaccine given at 10 to 12 months of age confers long-lasting immunity to 95% of vaccinees (16). The second dose, given to 6-year-olds, raises the conversion rate to nearly 100%, eliminating primary vaccine failures (45). The two-dose strategy has been credited with elimination of indigenous measles in several countries (9), and the live attenuated MV vaccine is considered to be one of the safest and most cost-effective health tools available (28).MV is a nonsegmented negative-strand RNA virus replicating in the cytoplasm. Vaccine safety and efficacy are sustained by lack of recombination, lack of a DNA replication phase, established vaccine production methods, and effective distribution networks (2, 50). A reverse genetic system (37) allows generation of recombinant MV expressing heterologous proteins, including those of other pathogens (48). For this, coding regions are inserted between duplicated MV-specific gene start and gene end motifs that direct transcription by the viral RNAdependent RNA polymerase. These expression cassettes are named additional transcription units (ATUs). The genomic location of an ATU determines the amount of protein expressed due to the sequential attenuation of transcription at gene ends (6,18).Work towards developing recombinant MV with additional vaccine specificities has begun: genes from hepatitis B virus (HBV) (43), simian and human immunodeficiency viruses (24, 52), mumps virus (50), and West Nile virus (10) have been inserted into different positions in the MV genome and thus expressed at different levels. The immunogenicities of vectored MVs and, in one case, their vaccine efficacies have been characterized in rodent and primate animal models. An MV-based candidate vaccine protected interferon (IFN) receptor-deficient mice against West Nile virus challenge (10).The widely used HBV vaccine is based on Saccharomyces cerevisiae-expressed small surface antigen (hepatitis B surface antigen [HBsAg]) and has a three-dose schedule (22). This vaccine provides enduring and protective immunity, but compliance with this regimen is often low, and a long-desired global immunization is not in sight. To facilitate HBV eradication, alternatives have been proposed, including a two-dose scheme (1, 4). Replicating HBsAg-expressing viral vectors have also been generated: vaccinia virus (27, 44)-, varicellazoster virus (20, 42)-, adenovirus (25)-, and MV-based vectors (43) produce HBsAg to high levels and elicit protective antiHBsAg antibodies in animal models.

TheStep Trial showed that the MRKAd5 HIV-1 subtype B Gag/Pol/Nef vaccine did not protect men from HIV infection or reduce setpoint plasma viral RNA (vRNA) levels but, unexpectedly, it did modestly enhance susceptibility to HIV infection in adenovirus type 5 (Ad5)-seropositive, uncircumcised men. As part of the process to understand the results of the Step Trial, we designed a study to determine whether rhesus macaques chronically infected with a host-range mutant Ad5 (Ad5hr) and then immunized with a replication defective Ad5 SIVmac239 Gag/Pol/Nef vaccine were more resistant or susceptible to SIV infection than unimmunized rhesus macaques challenged with a series of escalating dose penile exposures to SIVmac 251. The Ad5 SIV vaccine induced CD8 ؉ T cell responses in 70% of the monkeys, which is similar to the proportion of humans that responded to the vaccine in the Step Trial. However, the vaccine did not protect vaccinated animals from penile SIV challenge. At the lowest SIV exposure dose (10 3 50% tissue culture infective doses), 2 of 9 Ad5-seropositive animals immunized with the Ad5 SIV vaccine became infected compared to 0 of 34 animals infected in the other animal groups (naive animals, Ad5-seropositive animals immunized with the empty Ad5 vector, Ad5-seronegative animals immunized with the Ad5 SIV vaccine, and Ad5-seronegative animals immunized with the empty Ad5 vector). Penile exposure to more concentrated virus inocula produced similar rates of infection in all animal groups. Although setpoint viral loads were unaffected in Step vaccinees, the Ad5 SIV-immunized animals had significantly lower acute-phase plasma vRNA levels compared to unimmunized animals. Thus, the results of the nonhuman primate (NHP) study described here recapitulate the lack of protection against HIV acquisition seen in the Step Trial and suggest a greater risk of infection in the Ad5-seropositive animals immunized with the Ad5 SIV vaccine. Further studies are necessary to confirm the enhancement of virus acquisition and to discern associated mechanisms.

Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever) in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis) as well as upon contact (by serum-dependent adhesion and phagocytosis). This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores.