Intranasal H5N1 vaccines, adjuvanted with chitosan derivatives, protect ferrets against highly pathogenic influenza intranasal and intratracheal challenge

We investigated the protective efficacy of two intranasal chitosan (CSN and TM-CSN) adjuvanted H5N1 Influenza vaccines against highly pathogenic avian Influenza (HPAI) intratracheal and intranasal challenge in a ferret model. Six groups of 6 ferrets were intranasally vaccinated twice, 21 days apart, with either placebo, antigen alone, CSN adjuvanted antigen, or TM-CSN adjuvanted antigen. Homologous and intra-subtypic antibody cross-reacting responses were assessed. Ferrets were inoculated intratracheally (all treatments) or intranasally (CSN adjuvanted and placebo treatments only) with clade 1 HPAI A/Vietnam/1194/2004 (H5N1) virus 28 days after the second vaccination and subsequently monitored for morbidity and mortality outcomes. Clinical signs were assessed and nasal as well as throat swabs were taken daily for virology. Samples of lung tissue, nasal turbinates, brain, and olfactory bulb were analysed for the presence of virus and examined for histolopathological findings. In contrast to animals vaccinated with antigen alone, the CSN and TM-CSN adjuvanted vaccines induced high levels of antibodies, protected ferrets from death, reduced viral replication and abrogated disease after intratracheal challenge, and in the case of CSN after intranasal challenge. In particular, the TM-CSN adjuvanted vaccine was highly effective at eliciting protective immunity from intratrache

Optimal inferred frequencies at high and low reassortment rates.

<p>Viral allele frequencies are shown for the example subjects as black dots. The optimised fit to the data, based on the assumption of a consistent fitness landscape across all subjects, is shown as a red dotted line, for the case of rapid reassortment between segments, and as a blue dotted line for the case of no reassortment between segments. Limiting reassortment between segments allows for the possibility of linkage disequilibrium between alleles on different segments, fitting observed correlations in allele frequencies. Data are shown for all polymorphic alleles at loci included in the model for the subjects <b>A.</b> Flu5001 and <b>B.</b> Flu013.</p

Challenge study subjects.

<p>Challenge study subjects.</p

Inferred haplotype fitnesses.

<p>Reported haplotypes show the composition of the viral sequence at the nucleotide positions HA 516, HA 1258, NP 327 and PA 1680 respectively. Colour indicates inferred relative fitness from blue (0) to red (1). The haplotypes form a hypercube; lines indicate haplotypes differing by a single nucleotide.</p

Individual-specific reassortment rates.

<p><b>A.</b> Optimal reassortment rates inferred for individual subjects. Error bars, shown as horizontal lines, correspond to confidence intervals obtained via a likelihood ratio test. <b>B.</b> Correlation between the extent of evidence for a limited reassortment rate, normalised by the extent of available data, and the size of the inoculum dose. The black line shows a linear regression model fitted to the data.</p

Single-locus scan for alleles potentially under selection.

<p>Bayesian Information Criterion (BIC) differences between the best selected model and the neutral model at each locus in the genome. Differences are reported for loci at which polymorphism was detected. A positive BIC difference indicates a weight of evidence in favour of selection, calculated using a single-locus model, applied to data from a single individual. Loci with a positive BIC difference are highlighted with vertical red dotted lines. Solid red vertical lines show loci at which selection was later identified using a multi-locus model with data from all individuals. Circles denote results from individuals receiving standard treatment; squares denote results from individuals receiving early treatment.</p

Patterns of allele frequency change are altered by the presence or absence of reassortment.

<p><b>A.</b> Representation of a three-locus system with large population size, and in which rapid reassortment wipes out linkage disequilibrium between alleles at different loci. The ‘A’ variant (red) is under mild negative selection, the ‘G’ variant (green) is under strong positive selection, while the ‘C’ variant is under weak positive selection. <b>B.</b> Given the rapid rate of reassortment, alleles at each locus evolve deterministically over time in accordance with the selection acting upon them. <b>C.</b> Representation of the same system, now with no reassortment. Alleles now evolve according the changes in the frequencies of full sequences in the population; each horizontal row indicates a set of sequences with one of four distinct three-locus genotypes. <b>D.</b> While the initial state of the system, and magnitudes of selection are unchanged, the fates of the red and blue variants are reversed. The AGT genotype is the fittest of the four genotypes; in a process known as genetic hitchhiking, the deleterious red variant is carried by the green to fixation. In a process known as clonal interference, the beneficial blue variant is wiped out in the competition. <b>E.</b> Representation of the same system in which there is finite, but limited reassortment between the variants. <b>F.</b> Given limited reassortment, the association between the red and green variants is gradually eroded so that the red variant is lost over time. After an initial decline, formation of the maximally fit TGC haplotype leads to the blue variant increasing in frequency over time.</p

Inferred reassortment rate across all individuals.

<p>BIC values from the MGML model, relative to the optimal value, for the early-treatment, standard-treatment, and combined datasets. Horizontal dashed lines indicate the relative BIC value given rapid reassortment between segments. For each dataset, a reassortment rate of zero was inferred, a higher BIC indicating a worse fit to the data. For scale we note that a difference of 10 BIC units indicates strong support in favour of a model.</p

Inferred fitness coefficients for the single-segment, multi-locus model across all individuals.

<p>Best fitting models are shown for each number of parameters; the best fitting model for data from each segment is highlighted in bold text. The parameter χ denotes an epistatic interaction between alleles.</p