Adenovirus F protein as a delivery vehicle for botulinum B.

BACKGROUND: Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. RESULTS: The Hcbetatre serotype B immunogen was genetically fused to Ad2F (Hcbetatre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcbetatre-Ad2F relative to Hcbetatre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcbetatre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcbetatre and Hcbetatre-Ad2F protected mice against 2.0 LD50. CONCLUSION: These results demonstrate that Hcbetatre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.

Transmission of single HIV-1 genomes and dynamics of early immune escape revealed by ultra-deep sequencing.

We used ultra-deep sequencing to obtain tens of thousands of HIV-1 sequences from regions targeted by CD8+ T lymphocytes from longitudinal samples from three acutely infected subjects, and modeled viral evolution during the critical first weeks of infection. Previous studies suggested that a single virus established productive infection, but these conclusions were tempered because of limited sampling; now, we have greatly increased our confidence in this observation through modeling the observed earliest sample diversity based on vastly more extensive sampling. Conventional sequencing of HIV-1 from acute/early infection has shown different patterns of escape at different epitopes; we investigated the earliest escapes in exquisite detail. Over 3-6 weeks, ultradeep sequencing revealed that the virus explored an extraordinary array of potential escape routes in the process of evading the earliest CD8 T-lymphocyte responses--using 454 sequencing, we identified over 50 variant forms of each targeted epitope during early immune escape, while only 2-7 variants were detected in the same samples via conventional sequencing. In contrast to the diversity seen within epitopes, non-epitope regions, including the Envelope V3 region, which was sequenced as a control in each subject, displayed very low levels of variation. In early infection, in the regions sequenced, the consensus forms did not have a fitness advantage large enough to trigger reversion to consensus amino acids in the absence of immune pressure. In one subject, a genetic bottleneck was observed, with extensive diversity at the second time point narrowing to two dominant escape forms by the third time point, all within two months of infection. Traces of immune escape were observed in the earliest samples, suggesting that immune pressure is present and effective earlier than previously reported; quantifying the loss rate of the founder virus suggests a direct role for CD8 T-lymphocyte responses in viral containment after peak viremia. Dramatic shifts in the frequencies of epitope variants during the first weeks of infection revealed a complex interplay between viral fitness and immune escape.

The impact of host immune status on the within-host and population dynamics of antigenic immune escape.

Antigenically evolving pathogens such as influenza viruses are difficult to control owing to their ability to evade host immunity by producing immune escape variants. Experimental studies have repeatedly demonstrated that viral immune escape variants emerge more often from immunized hosts than from naive hosts. This empirical relationship between host immune status and within-host immune escape is not fully understood theoretically, nor has its impact on antigenic evolution at the population level been evaluated. Here, we show that this relationship can be understood as a trade-off between the probability that a new antigenic variant is produced and the level of viraemia it reaches within a host. Scaling up this intra-host level trade-off to a simple population level model, we obtain a distribution for variant persistence times that is consistent with influenza A/H3N2 antigenic variant data. At the within-host level, our results show that target cell limitation, or a functional equivalent, provides a parsimonious explanation for how host immune status drives the generation of immune escape mutants. At the population level, our analysis also offers an alternative explanation for the observed tempo of antigenic evolution, namely that the production rate of immune escape variants is driven by the accumulation of herd immunity. Overall, our results suggest that disease control strategies should be further assessed by considering the impact that increased immunity--through vaccination--has on the production of new antigenic variants.