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Abstract Objective This manuscript aims to provide an overview of the unique considerations and best practice principles associated with the manufacture of human viral challenge agents. Results Considerations are discussed on the entire process from strain and viral source selection through manufacturing, safety and efficacy testing. The human viral challenge (HVC) model is an important tool to help accelerate the drug development process but producing viruses suitable for use in the model presents a unique set of challenges. There are many case by case decisions and risk assessments to consider and no clear international standard to produce viruses for this purpose. The authors present challenge virus manufacturing considerations from the current literature, regulatory guidance and their own direct experience in producing challenge viruses. The use of these viral stocks in clinical studies, as published in peer-reviewed journals, is also briefly described.

Abstract The Human Viral Challenge (HVC) model has, for many decades, helped in the understanding of respiratory viruses and their role in disease pathogenesis. In a controlled setting using small numbers of volunteers removed from community exposure to other infections, this experimental model enables proof of concept work to be undertaken on novel therapeutics, including vaccines, immunomodulators and antivirals, as well as new diagnostics. Crucially, unlike conventional phase 1 studies, challenge studies include evaluable efficacy endpoints that then guide decisions on how to optimise subsequent field studies, as recommended by the FDA and thus licensing studies that follow. Such a strategy optimises the benefit of the studies and identifies possible threats early on, minimising the risk to subsequent volunteers but also maximising the benefit of scarce resources available to the research group investing in the research. Inspired by the principles of the 3Rs (Replacement, Reduction and Refinement) now commonly applied in the preclinical phase, HVC studies allow refinement and reduction of the subsequent development phase, accelerating progress towards further statistically powered phase 2b studies. The breadth of data generated from challenge studies allows for exploration of a wide range of variables and endpoints that can then be taken through to pivotal phase 3 studies. We describe the disease burden for acute respiratory viral infections for which current conventional development strategies have failed to produce therapeutics that meet clinical need. The Authors describe the HVC model’s utility in increasing scientific understanding and in progressing promising therapeutics through development. The contribution of the model to the elucidation of the virus-host interaction, both regarding viral pathogenicity and the body’s immunological response is discussed, along with its utility to assist in the development of novel diagnostics. Future applications of the model are also explored.

We characterise the evolutionary dynamics of influenza infection described by viral sequence data collected from two challenge studies conducted in human hosts. Viral sequence data were collected at regular intervals from infected hosts. Changes in the sequence data observed across time show that the within-host evolution of the virus was driven by the reversion of variants acquired during previous passaging of the virus. Treatment of some patients with oseltamivir on the first day of infection did not lead to the emergence of drug resistance variants in patients. Using an evolutionary model, we inferred the effective rate of reassortment between viral segments, measuring the extent to which randomly chosen viruses within the host exchange genetic material. We find strong evidence that the rate of effective reassortment is low, such that genetic associations between polymorphic loci in different segments are preserved during the course of an infection in a manner not compatible with epistasis. Combining our evidence with that of previous studies we suggest that spatial heterogeneity in the viral population may reduce the extent to which reassortment is observed. Our results do not contradict previous findings of high rates of viral reassortment in vitro and in small animal studies, but indicate that in human hosts the effective rate of reassortment may be substantially more limited.

A Proteosome-adjuvanted trivalent inactivated influenza vaccine (P-TIV) administered intra-nasally was shown to be safe, well tolerated and immunogenic in both systemic and mucosal compartments, and effective at preventing illness associated with evidence of influenza infection.In two separate studies using the human viral challenge model, subjects were selected to be immunologically naive to A/Panama/2007/1999 (H3N2) virus and then dosed via nasal spray with one of three regimens of P-TIV or placebo. One or two doses, 15 μg or 30 μg, were given either once only or twice 14 days apart (1 x 30 μg, 2 x 30 μg, 2 x 15 μg) and subjects were challenged with A/Panama/2007/1999 (H3N2) virus. Immune responses to the vaccine antigens were measured by haemagglutination inhibition assay (HAI) and nasal wash secretory IgA (sIgA) antibodies.Vaccine reactogenicity was mild, predictable and generally consistent with earlier Phase I studies with this vaccine. Seroconversion to A/Panama/2007/1999 (H3N2), following vaccination but prior to challenge, occurred in 57% to 77% of subjects in active dosing groups and 2% of placebo subjects. The greatest relative rise in sIgA, following vaccination but prior to challenge, was observed in groups that received 2 doses.Intranasal vaccination significantly protected against influenza (as defined by influenza symptoms combined with A/Panama seroconversion) following challenge with A/Panama/2007/1999 (H3N2). When data were pooled from both studies, efficacy ranged from 58% to 82% in active dosing groups for any influenza symptoms with seroconversion, 67% to 85% for systemic or lower respiratory illness and seroconversion, and 65% to 100% for febrile illness and seroconversion. The two dose regimen was found to be superior to the single dose regimen. In this study, protection against illness associated with evidence of influenza infection (evidence determined by seroconversion) following challenge with virus, significantly correlated with pre-challenge HAI titres (p = 0.0003) and mucosal sIgA (p≤0.0001) individually, and HAI (p = 0.028) and sIgA (p = 0.0014) together. HAI and sIgA levels were inversely related to rates of illness.ClinicalTrials.gov NCT02522754.

Influenza is a major cause of morbidity and mortality. Despite vaccination, many elderly recipients do not develop a protective antibody response. To determine whether Human Leukocyte Antigen (HLA) alleles modulate seroprotection to influenza, a cohort of HLA class II-typed high-risk vaccine recipients was investigated. Haemagglutinin inhibition (HAI) titres were measured 14-40 days post-subunit vaccination. Seroprotection was defined as HAI titres reaching 40 or greater for all three vaccine strains. HLA-DRB1*04∶01 and HLA-DPB1*04∶01 alleles were detected at higher frequencies in seroprotected compared with non-seroprotected individuals. Thus, the presence of certain HLA class II alleles may determine the magnitude of antibody responses to influenza vaccination.